Reporter constructs for compound screening

ABSTRACT

The instant description provides reporter constructs, transgenic cells, and transgenic organisms and methods for identifying agents that can regulate gene expression and improve plant performance and yield. Compounds that increase plant performance or yield are identified by contacting a test compound with a plant cell that comprises a target promoter sequence operably linked to a polynucleotide sequence encoding a DNA sequence-specific transactivator, and a reporter polynucleotide that is operably linked to a promoter sequence that is recognized by the DNA sequence-specific transactivator. The target promoter sequence can be recognized by a transcriptional regulatory polypeptide capable of modulating specific signaling pathways that enhance plant performance or yield.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §§365 and 371, this application is a United StatesNational Stage Application that claims priority to InternationalApplication No. PCT/US2011/037573 filed on May 23, 2011 (expired), whichclaims the benefit of U.S. provisional patent application 61/347,516,filed on May 24, 2010. PCT application PCT/US2011/037573 is acontinuation of PCT application PCT/US2010/045941, filed on Aug. 18,2010 (expired). The entire contents of each of these applications arehereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to reporter constructs, transgenic cellsand transgenic organisms that can be used for the identification ofagents that regulate gene expression.

BACKGROUND OF THE INVENTION

Manipulation of organisms or in vitro cultures to alter and/or improvephenotypic characteristic often requires the modulation of geneexpression. For example, the stress tolerance of a plant can be improvedthrough modifying the expression of genes involved in signaltransduction pathways related to various stress responses. One way toachieve this goal is to genetically engineer the organisms or in vitrocultures, an approach that is costly and time consuming. An alternativeapproach is to identify chemical compounds that can be applied to theseorganisms, e.g., plants, mammals, yeast, Drosophila, C. elegans, orbacteria etc., or their in vitro cultures to obtain the desiredphenotypes. Currently, chemicals are screened through reporterconstructs in which a reporter polynucleotide is directly fused to apromoter sequence that is capable of being recognized by atranscriptional regulatory protein, i.e., proteins that can regulate thesignaling pathways that contribute to the development of a desiredtrait. However, this conventional approach has limited sensitivity thatleads to inefficient compound screening and/or requires significanteffort to identify reporter cell lines or organisms suitable for thescreen, i.e., reporter cell lines or organisms in which the function orexpression of the transcriptional regulatory protein can be altereddirectly or indirectly in response to a compound treatment so that theamount of the resulting reporter molecule is readily discernible fromthe controls.

The present specification provides, inter alia, novel vectors, celllines, and methods useful for modulating gene expression, identifyingand analyzing regulatory sequences, and discovering new targets andreagents for improving plant performance or therapeutic intervention inhuman disease. The reporter constructs of the specification containarrangements of additional genetic elements that can optimize thesignal-to-noise ratio of the conventional promoter-reporter assay,minimize the efforts on characterization and development of reporterlines, and thus improve the sensitivity and the efficiency of thescreens. These novel constructs and methods can also be used in ahigh-throughput format to identify agents that can be rapidly deployedinduce modified gene expression and/or desired phenotypic alterations inorganisms, for example, compounds can be applied to plants through aspray or via irrigation.

Examples of how to employ these reporter constructs and transgenic cellsand organisms to identify useful chemical compounds are provided. Otheraspects and embodiments of the specification are described below or canbe derived from the teachings of this disclosure as a whole.

SUMMARY OF THE INVENTION

The present description provides a novel series of constructs,transgenic cells, transgenic organisms and methods which permit theidentification of novel sequences and agents that are capable ofmodulating gene expression.

In one aspect, the present description provides a transcriptional fusionreporter system where a reporter gene construct comprises, in operablelinkage, a target promoter sequence that can be recognized by atranscription regulatory protein, a polynucleotide sequence that encodesa DNA sequence-specific transactivator, and a reporter gene. Thereporter gene expression is regulated by both the target promoter andthe DNA sequence-specific transactivator.

In another aspect, the instant description provides a translationalfusion reporter system where a reporter gene construct of the inventioncomprises, in operable linkage, a polynucleotide encoding a DNAsequence-specific transactivator and a polynucleotide encoding atranslational fusion of a reporter molecule and a polypeptide ofinterest. The DNA sequence-specific transactivator regulates theexpression of the translational fusion protein.

The DNA sequence-specific transactivator of the reporter gene constructof the invention contains at least a DNA binding domain and atranscription activation domain, for example, LEXA:GAL4, SEQ ID NO: 4 (atranslational fusion of the DNA binding domain of LEXA and theactivation domain of GAL4), or GAL4:VP16, SEQ ID NO: 28 (a translationalfusion of the DNA binding domain of GAL4 and the transcription activatorprotein VP16).

In some embodiments, the DNA sequence-specific transactivator is asteroid-inducible transactivator, which regulates transcription whenbound by a steroid, for example, LEXA:GAL4:GR, SEQ ID NO: 2 (atranslational fusion of the DNA binding domain of LEXA, the activationdomain of GAL4, and the ligand binding domain of glucocorticoidreceptor), or GAL4:VP16:GR, SEQ ID NO: 7 (a translational fusion of theDNA binding domain of GAL4, the transcription activator protein VP16,and the ligand binding domain of glucocorticoid receptor). Thesetransactivators remain in the cytoplasm until they bind dexamethasone, aglucocorticoid receptor agonist. The dexamethasone bound transactivatorsthen translocate into the nucleus and activate the expression of atarget protein, e.g. a reporter, a translational fusion of a reportermolecule and a polypeptide of interest, or a transcriptional regulatorypolypeptide that regulates the expression thereof.

In some embodiments, the reporter gene construct comprises a dual-twocomponent reporter system where a steroid-inducible DNA sequencespecific transactivator, such as GAL4:VP16:GR (SEQ ID NO: 7), binds asteroid, for example, dexamethasone, and activates the expression of atranscriptional regulatory polypeptide that recognizes a target promotersequence operably linked to a polynucleotide that encodes an additionalDNA sequence-specific transcriptional activator, for example, LEXA:GAL4(SEQ ID NO: 4). A reporter molecule or a fusion protein of a reportermolecule and a polypeptide of interest would be expressed from apromoter that can be recognized by said DNA sequence-specifictranscriptional activator, for example, the opLexA promoter (SEQ ID NO:5).

The reporter molecule of the instant description can be any reportergene molecule, for example, reporter gene molecules or reporterpolynucleotide whose expression or activity can be measured bycalorimetric, fluorescent or luminescence signals, such as greenfluorescent protein (GFP), luciferase (LUC), chloramphenicol transferase(CAT), and glucuronidase (GUS).

In another aspect, the instant description provides transgenic organismsor cells derived therefrom, such as microbes, mammals, yeast,Drosophila, C. elegans, which are transformed with the reporter geneconstructs as described above.

In yet another aspect, the instant description provides methods ofidentifying chemical compounds comprising the steps of contacting atleast one test compound with a cell that was transformed with any one ofthe reporter gene constructs as described above, and selecting acompound that alters the reporter gene activity relative to controls.

The instant description also provides compounds identified in accordancewith the methods.

The instant description also provides methods to enhance plantperformance by applying the identified compound to plants.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING AND DRAWINGS

The Sequence Listing provides exemplary polynucleotide and polypeptidesequences of the instant description.

Incorporation of the Sequence Listing.

The copy of the Sequence Listing, being submitted electronically withthis patent application, provided under 37 CFR §1.821-1.825, is aread-only memory computer-readable file in ASCII text format. TheSequence Listing is named “MBI-0096PCT_ST25.txt”. The electronic file ofthe Sequence Listing was created on May 23, 2011, and is 76,901 bytes insize measured in MS-WINDOWS. The Sequence Listing is herein incorporatedby reference in its entirety.

FIGS. 1-9 depict reporter assay systems that can be introduced into anorganism or a cell by transformation. The resulting transgenic organismor cell can be employed to identify useful chemistry that regulates asignaling pathway of interest. Ovals represent the polypeptides,rectangles represent polynucleotides, and hexagons represent one or morechemical compounds. Lines with arrow-heads indicate a direct or indirectchemical enhancement of activity whereas blunt-ended lines indicate adirect or indirect repression of activity by a chemical compound. Filledcircles represent dexamethasone. FUSION represents a translationalfusion of a polypeptide of interest and a reporter molecule, or itsencoding polynucleotide. DBD:AD represents a translational fusion of aDNA binding domain (DBD) and a transcriptional activation domain (AD),or its encoding polynucleotide. DBD site is a promoter sequence which isbound by the DBD. REPORTER is any fluorescent, colorimetric orluminescent reporter, e.g. GFP (SEQ ID NO: 8), GUS (SEQ ID NO: 9), orluciferase (SEQ ID NO: 10), or a polynucleotide that encodes any of theaforementioned reporters. prTARGET represents a promoter can berecognized by a transcriptional regulatory polypeptide in the instantdescription. PROMOTER represents any promoter sequence. Roman numeralsrepresent the serial events that may occur associated with the treatmentof the chemical compounds. DBD:AD:GR represents adexamethasone-inducible transactivator that encodes a translationalfusion of a DNA-binding domain (DBD), a transcriptional activationdomain (AD) and the ligand binding domain of the glucocorticoidreceptor. DBD can be any DNA binding domain, for example, the DNAbinding domain of GAL4, SEQ ID NO: 18, or the DNA binding domain ofLexA, SEQ ID NO: 16:AD represent any transcription activation domain,for example, the activation domain of GAL4, SEQ ID NO: 20; VP16, SEQ IDNO: 22; EDLL, SEQ ID NO: 23-26.

FIG. 1. Transcriptional fusion assay system I: i) transcription andtranslation of a DNA sequence-specific transcriptional activatorDBD:AD:GR; ii) translocation of the DNA sequence-specifictranscriptional activator into the nucleus upon addition ofdexamethasone, and binding to its cognate promoter sequence DBD site;iii) transcription and translation of the reporter (reported); iv)transcription and translational of a transcriptional regulatorypolypeptide leads to v) binding of the transcriptional regulatorypolypeptide to its target promoter sequence; vi) modulation of thetranscriptional regulatory polypeptide binding or activation by acompound through direct or indirect mechanisms; vii) transcription andtranslation of the reporter (reporter2).

FIG. 2. Transcriptional fusion assay system II: i) compound-mediatedinduction or repression of DBD:AD transcription under the regulatorycontrol of a promoter recognized by a transcriptional regulatorypolypeptide; ii) transcription/translation of DBD:AD; (iii) binding andactivating the DBD site promoter; (iv) transcription/translation of thereporter gene.

FIG. 3. Transcriptional fusion assay system III: i) transcription andtranslation of DBD1:AD1:GR; ii) translocation of DBD1:AD1:GR into thenucleus with binding to the DBD1 site upon the addition ofdexamethasone; iii) transcription and translation of the reporter(reported); iv) transcription and translational of the targettranscriptional regulatory polypeptide, which leads to v) binding of thetranscriptional regulatory polypeptide to the target promoter sequence;vi) modulation of transcriptional regulatory polypeptide binding oractivation through direct or indirect mechanisms by a chemical compound;vii) transcription/translation of DBD2:AD; viii) binding of DBD2: AD tothe DBD2site promoter; ix) transcription and translation of the reporter(reporter2).

FIG. 4. Translational fusion assay system I: i)transcription/translation of DBD:AD; ii) binding to and activation ofthe DBD site promoter; iii) transcription/translation of the fusionprotein comprising of a reporter molecule and a polypeptide of interest;iv) stabilization or degradation of the fusion protein upon thetreatment of chemical compounds.

FIG. 5. Translational fusion assay system II. Two rounds ofamplification. i) transcription/translation of DBD1:AD; ii) binding toand activation of the DBD1 site promoter; iii) transcription/translationof a second DNA sequence-specific transcriptional activator DBD2:AD; iv)binding and activating the DBD2 site promoter; v)transcription/translation of the fusion protein comprising of a reportermolecule and a polypeptide of interest; vi) stabilization or degradationof the fusion protein upon the treatment of chemical compounds.

FIG. 6. A two-component transcriptional fusion system of the instantdescription showed increased signal-to-noise ratio compared to aone-component transcriptional fusion system. Cell lines transformed withA (a direct promoter GFP transcriptional fusion), i.e., “Z27379”, andcell lines transformed with B (a two-component transcription fusionreporter system II), i.e., “Z164567”, “Z164569”, “Z164571”, “Z164572”.“Z164577”, “Z164578”, “Z164581”, “Z164583”, “Z164588”, “Z164589”, weresubjected to an induction treatment, which activates the prTargetpromoter (black columns), or a mock treatment (gray columns) before dataacquisition. The respective GFP fluorescence values are indicated by theY axis and the standard errors are shown at the top of the columns. Thenumbers on top of some columns represent the fold of reporter inductionof the induction treatment over the mock treatment.

FIG. 7. Compound screening using the direct fusion and the two-componenttranscriptional fusion systems. Cell lines transformed withprTARGET::GFP (the direct fusion reporter system) orprTARGET::LexA:Gal4_oplexA::GFP (the two-component reporter system) wereused in a primary screen on a 30K diverse compound panel to identifycompounds that can induce the prTARGET promoter, Fold of induction,calculated by the GFP florescence value of the test compound-treatedgroup relative to that of the control (DMSO)-treated group, isrepresented on the X axis for two-component system, and on the Y axisfor the direct fusion reporter system. Several compounds that did notmeet the threshold of 2.5 fold induction with the direct fusion reporterline were identified as hits using the improved two-component system,demonstrating the improved sensitivity of the two-component system.

FIG. 8. Four (4) out of twenty (20) compound hits, identified from aprimary screen using the two-component reporter line, “C71125”,“C66433”, “C71126” and “C71124”, conferred significant tolerance todesiccation stress with Arabidopsis seedlings relative to controlcompound, DMSO.

FIG. 9. A two-component transcriptional fusion system comprising astress-inducible promoter RD29A. Cell lines transformed withprRD29A::LEXA:GAL4_opLEXA::GFP, i.e., “1-3-5”, “5-3-1”, “6-8-5”,“8-2-2”. “4-7-3”, “2-7-1”, and cell lines transformed with prRD29A::GFP(i.e., “RD29A”) or prRD29B::GFP, (i.e., “RD29B”), were subjected to anABA induction (black columns), or a mock treatment (gray columns) beforedata acquisition. The relative GFP fluorescence values are indicated bythe Y axis and the standard errors are shown at the top of the columns.

DETAILED DESCRIPTION

The instant description relates generally to reporter constructs andtheir use in gene regulation. The instant description provides methodsfor identification of chemical compounds that can be applied to enhancethe performance or modify phenotypes of an organism or in vitro culture.Throughout this disclosure, various information sources are referred toand/or are specifically incorporated. The information sources includescientific journal articles, patent documents, textbooks, and World WideWeb browser-inactive page addresses. While the reference to theseinformation sources clearly indicates that they can be used by one ofskill in the art, each and every one of the information sources citedherein are specifically incorporated in their entirety, whether or not aspecific mention of “incorporation by reference” is noted. The contentsand teachings of each and every one of the information sources can berelied on and used to make and use embodiments of the instantdescription.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include the plural reference unless the context clearlydictates otherwise. Thus, for example, a reference to “a host cell”includes a plurality of such host cells, and a reference to “a stress”is a reference to one or more stresses and equivalents thereof known tothose skilled in the art, and so forth.

DEFINITIONS

“Nucleic acid molecule” refers to an oligonucleotide, polynucleotide orany fragment thereof. It may be DNA or RNA of genomic or syntheticorigin, double-stranded or single-stranded, and combined withcarbohydrate, lipids, protein, or other materials to perform aparticular activity such as transformation or form a useful compositionsuch as a peptide nucleic acid (PNA).

“Polynucleotide” is a nucleic acid molecule comprising a plurality ofpolymerized nucleotides, e.g., at least about 15 consecutive polymerizednucleotides. A polynucleotide may be a nucleic acid, oligonucleotide,nucleotide, or any fragment thereof. In many instances, a polynucleotidecomprises a nucleotide sequence encoding a polypeptide (or protein) or adomain or fragment thereof. Additionally, the polynucleotide maycomprise a promoter, an intron, an enhancer region, a polyadenylationsite, a translation initiation site, 5′ or 3′ untranslated regions, areporter gene, a selectable marker, or the like. The polynucleotide canbe single-stranded or double-stranded DNA or RNA. The polynucleotideoptionally comprises modified bases or a modified backbone. Thepolynucleotide can be, e.g., genomic DNA or RNA, a transcript (such asan mRNA), a cDNA, a PCR product, a cloned DNA, a synthetic DNA or RNA,or the like. The polynucleotide can be combined with carbohydrate,lipids, protein, or other materials to perform a particular activitysuch as transformation or form a useful composition such as a peptidenucleic acid (PNA). The polynucleotide can comprise a sequence in eithersense or antisense orientations. “Oligonucleotide” is substantiallyequivalent to the terms amplimer, primer, oligomer, element, target, andprobe and is preferably single-stranded.

A “recombinant polynucleotide” is a polynucleotide that is not in itsnative state, e.g., the polynucleotide comprises a nucleotide sequencenot found in nature, or the polynucleotide is in a context other thanthat in which it is naturally found, e.g., separated from nucleotidesequences with which it typically is in proximity in nature, or adjacent(or contiguous with) nucleotide sequences with which it typically is notin proximity. For example, the sequence at issue can be cloned into avector, or otherwise recombined with one or more additional nucleicacid.

An “isolated polynucleotide” is a polynucleotide, whether naturallyoccurring or recombinant, that is present outside the cell in which itis typically found in nature, whether purified or not. Optionally, anisolated polynucleotide is subject to one or more enrichment orpurification procedures, e.g., cell lysis, extraction, centrifugation,precipitation, or the like.

A “reporter polynucleotide” is a polynucleotide that encodes a reporterprotein, whose expression level or activity can be quantified bycolorimetric, fluorescent or luminescence signals. Commonly-usedreporter proteins include but are not limited to, beta-galactosidase(LacZ), green fluorescent protein (GFP), luciferase (LUC),chloramphenicol transferase (CAT), and glucuronidase (GUS).

“Gene” or “gene sequence” refers to the partial or complete codingsequence of a gene, its complement, and its 5′ or 3′ untranslatedregions. A gene is also a functional unit of inheritance, and inphysical terms is a particular segment or sequence of nucleotides alonga molecule of DNA (or RNA, in the case of RNA viruses) involved inproducing a polypeptide chain. The latter may be subjected to subsequentprocessing such as chemical modification or folding to obtain afunctional protein or polypeptide. A gene may be isolated, partiallyisolated, or found with an organism's genome. By way of example, atranscriptional regulatory polypeptide gene encodes a transcriptionalregulatory polypeptide, which may be functional or require processing tofunction as an initiator of transcription.

Operationally, genes may be defined by the cis-trans test, a genetictest that determines whether two mutations occur in the same gene andthat may be used to determine the limits of the genetically active unit(Rieger et al. (1976)^(i)). A gene generally includes regions preceding(“leaders”; upstream) and following (“trailers”; downstream) the codingregion. A gene may also include intervening, non-coding sequences,referred to as “introns”, located between individual coding segments,referred to as “exons”. Most genes have an associated promoter region, aregulatory sequence 5′ of the transcription initiation codon (there aresome genes that do not have an identifiable promoter). The function of agene may also be regulated by enhancers, operators, and other regulatoryelements.

A “transgenic or transformed plant” refers to a plant which contains arecombinant polynucleotide introduced by transformation. Transformationmeans introducing a nucleotide sequence in a plant in any manner tocause stable or transient expression of the sequence. This may beachieved by transfection with viral vectors, transformation withplasmids, such as Agrobacterium-based vectors, or introduction of nakedDNA by electroporation, lipofection, or particle gun acceleration. Atransformed plant may refer to a whole plant as well as to seed, planttissue, plant cells or any other plant material, and to the plant'sprogeny.

A “transgenic organism” refers to an organism, such as a plant, amicrobe, a mammal, yeast, Drosophila, C. elegans, etc., which contains arecombinant polynucleotide introduced by transformation. Transformationmeans introducing a nucleotide sequence in an organism in any manner tocause stable or transient expression of the sequence. This may beachieved by transfection with viral vectors, transformation withplasmids, or introduction of naked DNA by electroporation, lipofection,or particle gun acceleration. A transformed organism may refer to awhole organism, to any part of the organism, to any materials derivedfrom the organism, and to an offspring of the organism.

A “vector” is a nucleic acid construct, generated recombinantly orsynthetically, comprising nucleic acid elements that can causeexpression of a gene. A “donor vector” is a construct for expression ofa polynucleotide sequence for a transactivator gene. The transactivatorgene is operably linked to a promoter. The promoter region may includetissue active-or-specific promoters, developmental stageactive-or-specific promoters, inducible promoters or constitutivepromoters.

A “polypeptide of interest” maybe any peptide, including, for example, apolypeptide sequence for a regulatory gene such as a transcriptionalregulatory polypeptide, a protein kinase or a phosphatase. Thesesequences may be in a sense or antisense orientation, or partial orcomplete gene sequences.

The phrase “altered or modified expression” in reference topolynucleotide or polypeptide expression refers to an expression patternin a transgenic organism that is different from the expression patternin the wild type plant or a reference plant; for example, by expressionin a cell type other than a cell type in which the sequence is expressedin the wild type plant, or by expression at a time other than at thetime the sequence is expressed in the wild type plant, or by a responseto different inducible agents, such as hormones or environmentalsignals, or at different expression levels (either higher or lower)compared with those found in a wild type plant. The term also refers tolowering the levels of expression to below the detection level orcompletely abolishing expression. The resulting expression pattern maybe transient or stable, constitutive or inducible.

A “promoter” or “promoter region” refers to an RNA polymerase bindingsite on a segment of DNA, generally found upstream or 5′ relative to acoding sequence under the regulatory control of the promoter. Thepromoter will generally comprise response elements that are recognizedby transcriptional regulatory polypeptides. transcriptional regulatorypolypeptide may bind to the promoter sequences, recruiting RNApolymerase, which synthesizes RNA from the coding region.Dissimilarities in promoter sequences account for different efficienciesof transcription initiation and hence different relative expressionlevels of different genes.

“Promoter function” includes regulating expression of the codingsequences under a promoter's control by providing a recognition site forRNA polymerase and/or other factors, such as transcriptional regulatorypolypeptides, all of which are necessary for the start of transcriptionat a transcription initiation site. A “promoter function” may alsoinclude the extent to which a gene coding sequence is transcribed to theextent determined by a promoter sequence.

The term “operably linked” refers to the association of polynucleotidesequences so that the function of one is affected by the other. Forexample, a promoter is operably linked with a coding sequence when itaffects the expression of that coding sequence (i.e., that the codingsequence is under the transcriptional control of the promoter). Codingsequences can be operably linked to regulatory sequences in sense orantisense orientation. The polynucleotide molecules may be part of asingle contiguous polynucleotide molecule and may be adjacent. Forexample, a promoter is operably linked to a gene of interest if thepromoter modulates transcription of the gene of interest in a cell.

The term “DNA sequence-specific transactivator” refers to a polypeptidethat comprises at least a DNA binding domain that binds to DNA with somedegree of specificity and a transcriptional activation domain that hasthe function of activating transcription. A common feature of someactivation domains is that they are designed to form amphiphilic alphahelices with negative charge (Giniger and Ptashne (1987) Nature330:670-672, Gill and Ptashne (1987) Cell 51:121-126, Estruch et al(1994) Nucl. Acids Res. 22:3983-3989). Examples include thetranscription activation region of VP16 or GAL4 (Moore et al. (1998)Proc. Natl. Acad. Sci. USA 95: 376-381; and Aoyama et al. (1995) PlantCell 7:1773-1785), peptides derived from bacterial sequences (Ma andPtashne (1987) Cell 51; 113-119) and synthetic peptides (Giniger andPtashne, supra), or the EDLL domain from plants (Mendel's PCTapplication PCT/US2009/048814). Exemplary transactivators are thosedescribed in Brent and Ptashne, U.S. Pat. No. 4,833,080, hereinincorporated by reference or in Hasselhoff and Hodge, WO97/30164.

“Activation” of a promoter-reporter construct is considered to beachieved when the activity value relative to control, e.g., a samplethat is not treated with a test compound, is 105%, 110%, 120%, 130%,140%, 150%, 200%, 250%, 300%, 400%, 500%, or 1000-3000% or more higher.

The phrases “coding sequence,” “structural sequence,” and “transcribablepolynucleotide sequence” refer to a physical structure comprising anorderly arrangement of nucleic acids. The nucleic acids are arranged ina series of nucleic acid triplets that each form a codon. Each codonencodes for a specific amino acid. Thus the coding sequence, structuralsequence, and transcribable polynucleotide sequence encode a series ofamino acids forming a protein, polypeptide, or peptide sequence. Thecoding sequence, structural sequence, and transcribable polynucleotidesequence may be contained, without limitation, within a larger nucleicacid molecule, vector, etc. In addition, the orderly arrangement ofnucleic acids in these sequences may be depicted, without limitation, inthe form of a sequence listing, figure, table, electronic medium, etc.

A “polypeptide” is an amino acid sequence comprising a plurality ofconsecutive polymerized amino acid residues e.g., at least about 15consecutive polymerized amino acid residues. In many instances, apolypeptide comprises a polymerized amino acid residue sequence that isa transcriptional regulatory polypeptide or a domain or portion orfragment thereof. Additionally, the polypeptide may comprise: (i) alocalization domain; (ii) an activation domain; (iii) a repressiondomain; (iv) an oligomerization domain; (v) a protein-proteininteraction domain; (vi) a DNA-binding domain; or the like. Thepolypeptide optionally comprises modified amino acid residues, naturallyoccurring amino acid residues not encoded by a codon, non-naturallyoccurring amino acid residues.

“Protein” refers to an amino acid sequence, oligopeptide, peptide,polypeptide or portions thereof whether naturally occurring orsynthetic.

A “recombinant polypeptide” is a polypeptide produced by translation ofa recombinant polynucleotide. A “synthetic polypeptide” is a polypeptidecreated by consecutive polymerization of isolated amino acid residuesusing methods well known in the art. An “isolated polypeptide,” whethera naturally occurring or a recombinant polypeptide, is more enriched in(or out of) a cell than the polypeptide in its natural state in awild-type cell, e.g., more than about 5% enriched, more than about 10%enriched, or more than about 20%, or more than about 50%, or more,enriched, i.e., alternatively denoted: 105%, 110%, 120%, 150% or more,enriched relative to wild type standardized at 100%. Such an enrichmentis not the result of a natural response of a wild-type organism.Alternatively, or additionally, the isolated polypeptide is separatedfrom other cellular components with which it is typically associated,e.g., by any of the various protein purification methods herein.

The instant description also encompasses production of DNA sequencesthat encode polypeptides and derivatives, or fragments thereof, entirelyby synthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents well known in the art. Moreover, syntheticchemistry may be used to introduce mutations into a sequence encodingpolypeptides or any fragment thereof.

“Derivative” refers to the chemical modification of a nucleic acidmolecule or amino acid sequence. Chemical modifications can includereplacement of hydrogen by an alkyl, acyl, or amino group orglycosylation, pegylation, or any similar process that retains orenhances biological activity or lifespan of the molecule or sequence.

The term “plant” includes whole plants, shoot vegetativeorgans/structures (for example, leaves, stems and tubers), roots,flowers and floral organs/structures (for example, bracts, sepals,petals, stamens, carpels, anthers and ovules), seed (including embryo,endosperm, and seed coat) and fruit (the mature ovary), plant tissue(for example, vascular tissue, ground tissue, and the like) and cells(for example, guard cells, egg cells, and the like), and progeny ofsame. The class of plants that can be used in the method of the instantdescription is generally as broad as the class of higher and lowerplants amenable to transformation techniques, including angiosperms(monocotyledonous and dicotyledonous plants), gymnosperms, ferns,horsetails, psilophytes, lycophytes, bryophytes, and multicellularalgae.

A “trait” refers to a physiological, morphological, biochemical, orphysical characteristic of a plant or particular plant material or cell.In some instances, this characteristic is visible to the human eye, suchas seed or plant size, or can be measured by biochemical techniques,such as detecting the protein, starch, or oil content of seed or leaves,or by observation of a metabolic or physiological process, e.g. bymeasuring tolerance to water deprivation or particular salt or sugarconcentrations, or by the observation of the expression level of a geneor genes, e.g., by employing Northern analysis, RT-PCR, microarray geneexpression assays, or reporter gene expression systems, or byagricultural observations such as hyperosmotic stress tolerance oryield. Any technique can be used to measure the amount of, comparativelevel of, or difference in any selected chemical compound ormacromolecule in the transgenic plants, however.

“Trait modification” refers to a detectable difference in acharacteristic in an organism being treated with the chemical compoundsof the instant description relative to a control organism of the samespecies, the latter including organisms treated with a control compoundor a carrier solvent or no treatment. In some cases, the traitmodification can be evaluated quantitatively. For example, the traitmodification can entail at least about a 2% increase or decrease, or aneven greater difference, in an observed trait as compared with a controlor wild-type organism. It is known that there can be a natural variationin the modified trait. Therefore, the trait modification observedentails a change of the normal distribution and magnitude of the traitin the plants as compared to control or wild-type organisms.

“Modulates” refers to a change in activity (biological, chemical, orimmunological) or lifespan resulting from specific binding between amolecule and either a nucleic acid molecule or a protein.

“Ectopic expression or altered expression or modified expression” inreference to a polynucleotide indicates that the pattern of expressionin, e.g., a transgenic organism or tissue, is different from theexpression pattern in a wild-type organism or a reference organism ofthe same species. The pattern of expression may also be compared with areference expression pattern in a wild-type plant of the same species.For example, the polynucleotide or polypeptide is expressed in a cell ortissue type other than a cell or tissue type in which the sequence isexpressed in the wild-type organism, or by expression at a time otherthan at the time the sequence is expressed in the wild-type plant, or bya response to different inducible agents, such as hormones orenvironmental signals, or at different expression levels (either higheror lower) compared with those found in a wild-type organism. The termalso refers to altered expression patterns that are produced by loweringthe levels of expression to below the detection level or completelyabolishing expression. The resulting expression pattern can be transientor stable, constitutive or inducible. In reference to a polypeptide, theterm “ectopic expression or altered expression” further may relate toaltered activity levels resulting from the interactions of thepolypeptides with exogenous or endogenous modulators or frominteractions with factors or as a result of the chemical modification ofthe polypeptides.

The term “overexpression” as used herein refers to a greater expressionlevel of a gene in an organism, a cell or a tissue, compared toexpression in a wild-type plant, cell or tissue, at any developmental ortemporal stage for the gene. Overexpression can occur when, for example,the genes encoding one or more polypeptides are under the control of astrong promoter (e.g., the cauliflower mosaic virus 35S transcriptioninitiation region). Overexpression may also occur under the control ofan inducible or tissue specific promoter. Thus, overexpression may occurthroughout an organism, in specific tissues of the organism, or in thepresence or absence of particular environmental signals, depending onthe promoter used.

Overexpression may take place in cells normally lacking expression ofpolypeptides functionally equivalent or identical to the presentpolypeptides. Overexpression may also occur in cells where endogenousexpression of the present polypeptides or functionally equivalentmolecules normally occurs, but such normal expression is at a lowerlevel. Overexpression thus results in a greater than normal production,or “overproduction” of the polypeptide in the organism, cell or tissue.

The term “transcription regulating region” or “transcription regulatingnucleic acid sequence” refers to a DNA regulatory sequence thatregulates expression of one or more genes in an organism when atranscriptional regulatory polypeptide having one or more specificbinding domains binds to the DNA regulatory sequence. Transcriptionalregulatory polypeptides possess a conserved domain. The transcriptionalregulatory polypeptides also comprise an amino acid subsequence thatforms a transcription activation domain that regulates expression of oneor more abiotic stress tolerance genes in an organism when thetranscriptional regulatory polypeptide binds to the regulating region.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The instant description provides novel reporter gene constructs thatcould be used to study gene regulation and identify novel sequences andagents that can be used to modify a phenotype of interest. The reportergene system of the instant description can be a transcriptional fusionreporter system or a translational fusion reporter system.

Transcriptional Fusion Reporter System

The transcriptional fusion reporter system of the instant descriptioncomprises a reporter gene that is regulated by: 1) a target promotersequence recognized by a transcriptional regulatory polypeptide, and 2)a DNA sequence-specific transactivator.

The transcriptional fusion reporter system can be used to identifycompounds that modulate the activity of a target promoter. Plants orplant cells transformed with the transcriptional fusion reporterconstructs can be treated with test compounds or compound libraries, andreporter gene expression can be monitored. Alternatively, thetransformed organisms or cells containing the transcriptional fusionreporter constructs can be placed in a panel of microtiter wells and apanel of test compounds can be added to the cells, one compound to eachwell. A useful compound can be identified based on its ability togenerate an enhanced or decreased reporter signal relative to controlcompounds.

The target promoters suitable for use herein can be any promoter that isrecognized and regulated by a transcriptional regulatory polypeptide,including those that are constitutively active or those that areinducible or tissue enhanced or developmental-stage active promoters. Ina preferred embodiment, the target promoters are the ones that regulateplant trait development. The target promoters can be naturally derivedor synthetically made. The minimal promoter for use in syntheticpromoters can be from any promoter. The minimal promoter supports basaltranscription and typically comprises regulatory elements such as TATAAsequences. Exemplary minimal promoter regions can be from promoters suchas the cauliflower mosaic virus (CaMV) 35 S transcription initiationregion, and other transcription initiation regions from various genesknown to those of skill.

In some embodiments of the instant description, the target promoteractivates the expression of a DNA sequence-specific transactivator,which recognizes and binds to a specific transcriptional regulatorysequence and induces high level reporter gene expression. For example,the target promoter could drive the expression of a translational fusionof a DNA binding domain (e.g., the LexA DNA binding domain) and atranscriptional activation domain (e.g., the Gal4 transcriptionalactivation domain). A reporter molecule would be expressed from apromoter bound by the DNA binding domain (e.g., the opLexA promoter)such that activation of the target promoter would result in amplifiedexpression of the reporter gene mediated by the DNA sequence-specifictransactivator (e.g., LEXA:GAL4) (FIG. 2). This system thus enables moresensitive reporter detection compared to direct promoter-reporter fusionconstruct, and is particularly advantageous when employing a promoter ofa transcriptional regulatory polypeptide or for targets with lowexpression levels or when screening for compounds that can down-regulatethe activity of a target promoter or a transcriptional regulatorypolypeptide that recognizes the target promoter.

The DNA sequence-specific transactivator of the transcriptional fusionsystem can also comprise a dexamethasone responsive element, forexample, a target promoter could drive the expression of a translationalfusion of the LexA DNA binding domain, the Gal4 transcriptionalactivation domain and the ligand binding domain of the glucocorticoidreceptor (GR). An example of this inducible reporter system involvesintroducing a dexamethasone-responsive cassette, e.g. a polynucleotideencoding a fusion protein LEXA:GAL4:GR, and a transcriptional fusion ofa target promoter and a reporter gene into the cell, where the additionof dexamethasone results in high levels of expression of atranscriptional regulatory polypeptide, which would, in turn, activate atarget promoter and result in high reporter expression (FIG. 1). Thesedexamethasone inducible reporter systems can be internally validated bythe addition or withdrawal of dexamethasone and the expected reporter(e.g., the “reporter2” in FIG. 1) signal induction can be quantifiedprior to a high throughput screen. The benefit of this multi-componentsystem would be the ease of identifying a candidate line with stronginduction characteristics, eliminating lines with silencing or poorexpression due to the chromosomal integration site. The control over theactivation of the transgene by dexamethasone also minimizes the negativeinterference from constitutive expression of some transgenes.Furthermore, this dexamethasone-inducible system can achieve adjustablelevels of reporter gene expression: in the absence of dexamethasone,endogenous transcriptional regulatory polypeptide binds to the targetpromoter and activates reporter gene expression to a relatively lowlevel, while in the presence of dexamethasone, dexamethasone-inducibletransactivator activates the expression of the exogenous transcriptionalregulatory polypeptide, which in turn drives high level expression ofthe reporter gene; this system conveniently enables screening forchemistries that can repress (in the presence of dexamethansone) oractivate (in the absence of dexamethasone) transcription from a targetpromoter with one singular construct and selected transgenic line;

A variant of the transcriptional fusion reporter system employs twodifferent DNA sequence-specific transactivators with distinct DNAbinding sequence specificities to activate reporter gene expression: afirst DNA sequence-specific transactivator, in the presence ofdexamethasone, induces the expression of a transcriptional regulatorypolypeptide, which, in turn, binds its target promoter sequence andactivates a second DNA sequence-specific transactivator, whichsubsequently activates the reporter gene expression (FIG. 3). A systemas such assimilates advantages of both systems described above (and asshown in FIGS. 1 and 2) by incorporating an amplification component thatboosts the sensitivity of the screen and a characterization componentthat eases the identification of suitable cell lines with stronginduction characteristics.

In addition to compound screening, the transcriptional fusion reportersystem can also be used to characterize novel promoter elements orpromoter fragments in response to various environmental stimuli oractivation signals. Any novel promoter element of interest can be usedas a target promoter and incorporated in the transcriptional fusionreporter system, which is then introduced into the cells and monitoredfor the ability to affect reporter gene activity under conditions thatcan activate a control promoter. The promoter function of the noveltarget promoter elements can be evaluated based on the ability toactivate or repress the reporter gene activity relative to the controlpromoter sequence under the same conditions.

Translational Fusion Reporter System

The translational fusion reporter system of the present descriptioncomprises at least 1) a polynucleotide encoding a DNA sequence specifictransactivator, 2) a polynucleotide encoding a fusion protein, and 3) anucleotide sequence recognized by the DNA sequence specifictransactivator. The genetic elements of 1), 2) and 3) are arranged in away such that the DNA sequence-specific transactivator activates theexpression of a translational fusion of a reporter molecule and apolypeptide of interest.

The translational reporter system of the instant description can be usedto identify compounds that can modulate the stability of a polypeptideof interest. Test compounds and control compounds are applied to thecells transformed with the translational fusion constructs. Testcompounds that change the stability of polypeptides of interest can beidentified based on the altered reporter gene activity levels relativeto controls. Hit compounds can be applied to the organisms of interest,for example, plants, bacteria, cell cultures etc., and further validatedfor the ability to change the stability of the polypeptide of theinterest using biochemical approaches that are known in the art.

The polypeptide of interest may be any polypeptide, but is preferably aregulatory polypeptide, such as a transcriptional regulatorypolypeptide, a phosphatase or a protein kinase. The polypeptide ofinterest may be from any species, particularly plant species, in anaturally occurring form or from any source whether natural, synthetic,semi-synthetic or recombinant. The polypeptide sequences may alsoinclude fragments of the present amino acid sequences of a regulatorpolypeptide, in particular a fragment with biological activity. In onepreferred embodiment, the polypeptides of interest are all thetranscriptional regulatory polypeptides identified in a plant, such asthose identified in Arabidopsis thaliana. These transcriptionalregulatory polypeptides collectively control all gene expression inplants and thus control all plant traits.

The plant transcriptional regulatory polypeptides may belong to one ofthe following transcription factor families: the AP2 (APETALA2) domaintranscription factor family (Riechmann and Meyerowitz (1998) J. Biol.Chem. 379:633-646); the MYB transcription factor family (Martin andPaz-Ares, (1997) Trends Genet. 13:67-73); the MADS domain transcriptionfactor family (Riechmann and Meyerowitz (1997) J. Biol. Chem.378:1079-1101); the WRKY protein family (Ishiguro and Nakamura (1994)Mol. Gen. Genet. 244:563-571); the ankyrin-repeat protein family (Zhanget al. (1992) Plant Cell 4:1575-1588); the zinc finger protein (Z)family (Klug and Schwabe (1995) FASEB J. 9: 597-604); the homeobox (HB)protein family (Duboule (1994) Guidebook to the Homeobox Genes, OxfordUniversity Press); the CAAT-element binding proteins (Forsburg andGuarente (1989) Genes Dev. 3:1166-1178); the squamosa promoter bindingproteins (SPB) (Klein et al. (1996) Mol. Gen. Genet. 1996 250:7-16); theNAM protein family (Souer et al. (1996) Cell 85:159-170); the IAA/AUXproteins (Rouse et al. (1998) Science 279:1371-1373); the HLH/MYCprotein family (Littlewood et al. (1994) Prot. Profile 1:639-709); theDNA-binding protein (DBP) family (Tucker et al. (1994) EMBO J.13:2994-3002); the bZIP family of transcription factors (Foster et al.(1994) FASEB J. 8:192-200); the Box P-binding protein (the BPF-1) family(da Costa e Silva et al. (1993) Plant J. 4:125-135); the high mobilitygroup (HMG) family (Bustin and Reeves (1996) Prog. Nucl. Acids Res. Mol.Biol. 54:35-100); the scarecrow (SCR) family (Di Laurenzio et al. (1996)Cell 86:423-433); the GF14 family (Wu et al. (1997) Plant Physiol.114:1421-1431); the polycomb (PCOMB) family (Kennison (1995) Annu. Rev.Genet. 29:289-303); the teosinte branched (TEO) family (Luo et al.(1996) Nature 383:794-799; the ABI3 family (Giraudat et al. (1992) PlantCell 4:1251-1261); the triple helix (TH) family (Dehesh et al. (1990)Science 250:1397-1399); the EIL family (Chao et al. (1997) Cell89:1133-44); the AT-HOOK family (Reeves and Nissen (1990)) Journal ofBiological Chemistry 265:8573-8582); the S1FA family (Zhou et al. (1995)Nucleic Acids Res. 23:1165-1169); the bZIPT2 family (Lu and Ferl (1995)Plant Physiol. 109:723); the YABBY family (Bowman et al. (1999)Development 126:2387-96); the PAZ family (Bohmert et al. (1998) EMBO J.17:170-80); a family of miscellaneous (MISC) transcription factorsincluding the DPBF family (Kim et al. (1997) Plant J. 11:1237-1251) andthe SPF1 family (Ishiguro and Nakamura (1994) Mol. Gen. Genet.244:563-571); the golden (GLD) family (Hall et al. (1998) Plant Cell10:925-936); or any other class of protein that is capable of directlyor indirectly binding DNA and regulating the expression of a targetgene.

Other transcriptional regulatory polypeptides may be identified byscreening polynucleotide or polypeptide sequence databases, such asGenBank, using sequence alignment methods and homology calculations,such as those described in Altschul et al. (1994) Nature Genetics 6:119-129. For example, the NCBI Basic Local Alignment Search Tool(BLAST®) (Altschul et al. (1990) J. Mol. Biol. 215:403-410) is availablefrom several sources, including the National Center for BiotechnologyInformation (NCBI, Bethesda, Md., for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastp, tblastn andtblastx). Alternatively, a program that identifies particular sequencemotifs may be employed along with specific characteristic consensussequences, such as FIND PATTERN (GCG, Madison, Wis.).

Exemplary plant transcriptional regulatory polypeptides that can beemployed in the instant description include Gilmour et al. (1998) teachan Arabidopsis AP2 transcription factor, CBF1, which, when overexpressedin transgenic plants, increases plant freezing tolerance. For anotherexample, Bruce et al. (2000); and Borevitz et al. (2000) teach that thePAP2 gene and other genes in the MYB family control anthocyaninbiosynthesis through regulation of the expression of genes known to beinvolved in the anthocyanin biosynthetic pathway. Applicants' own patentpublication no. US20080010703A1, which is herein incorporated byreference in its entirety, has disclosed regulatory proteins involved inplant light signaling pathways and alteration in the expression oractivity of which have resulted in increased yield in plants. Inaddition, Applicants' patent publication no. US20090138981A1, which isherein incorporated by reference in its entirety, has listedtranscriptional regulatory polypeptides that are involved in variousplant signaling pathways and cellular events that impact on plantdisease resistance, biomass production and abiotic stress tolerance.Accordingly, one skilled in the art would recognize that changing theexpression of the present sequences in a plant would introduce modifiedtraits not found in the wild-type cultivar or strain.

The DNA sequence-specific transactivator can be any transactivator thatcomprises at least a DNA binding domain and a transcriptional activationdomain and has transcription-regulation activity, for example, atranslational fusion of the LexA DNA binding domain and the Gal4transcriptional activation domain. The translational fusion system ofthe instant description comprising DNA sequence-specific transactivatorenables high level of reporter expression can be especially useful foridentifying compounds that can decrease the stability of a polypeptideof interest (FIG. 4). Similar to what has been described in thetranscriptional reporter system described above, various arrangementsand combinations of sequences encoding DNA sequence-specifictransactivator and dexamethasone inducible cassettes can be incorporatedinto the system in order to facilitate reporter line characterizationand development, improve signal to noise ratio of the screening assay,and enhance screening efficiency. In addition, the various geneticelements included in both the transcriptional fusion reporter systemsand translational fusion reporter systems described herein may reside ina single construct, or in multiple constructs with various selectionmarkers. The multiple constructs can be transformed into an organism ofinterest, and transgenic organisms or cells carrying the desired geneticelements can be identified through the detection of the expression ofappropriate selection markers. For example, a reporter system of theinstant description may consist of two reporter constructs, with thepolynucleotide encoding a DNA sequence-specific transactivator, e.g.LEXA:GAL4, comprised in one construct and the promoter responsive to theDNA sequence-specific transactivator, e.g. opLEXA, and thepolynucleotide encoding the reporter or reporter fusion in the other.

Reporter Genes

Reporter genes suitable for use in the instant description are known tothose of skill in the art. Reporters can be any protein, and include,but are not limited to, fluorescent proteins, such as green or redfluorescent proteins, or variants that produce a fluorescent color;β-glucuronidase (GUS); luciferase; chloramphenicol acetyltransferase;β-galactosidase; and alkaline phosphatase. Commonly used reporter genesinclude those encoding proteins that can generate quantifiablefluorescent, colorimetric, or luminescent signals. Transcription of thesequences encoding the reporter gene can be determined using any methodknown in the art. In some embodiments, protein activity of the reportergene is measured, e.g., using a fluorescent reader or otherinstrumentation appropriate to the reporter system. Products to assistin determination of reporter activity are commercially available.

Samples that are treated with a test compound, or pool of testcompounds, are compared to control samples without the test compound toexamine the extent of modulation. Control samples (untreated withactivators are assigned a relative activity value. Activation is thenachieved when the reporter activity value relative to the control is105%, 105-150%, optionally 150%, 150-500%, or 500-2000% or more, whereasdown-regulation is achieved when the reporter activity value relative tothe control is 70-90%, 66%, 20-50%, or 5-10%.

In other embodiments, endpoints other than reporter activity areassayed. For example, expression levels of the mRNA or protein can bemeasured to assess the effects of a test compound on reporteractivation. In this instance, the expression of the reporter constructis measured by assessing the level of mRNA that encodes the reportergene or the translational fusion of the reporter gene and a polypeptideof interest, or alternatively of the protein product. These assays canbe performed using any methods known by those of skill in the art to besuitable. For example, mRNA expression can be detected usingamplification-based methodologies, northern or dot blots, nucleaseprotection and the like. Polypeptide products can be identified usingimmunoassays.

Introduction of Reporter Constructs into Hosts or Host Cells

Reporter constructs can be introduced into the desired hosts or cellsderived therefrom, such as plants, microbes, mammals, yeast, Drosophila,C. elegans by a variety of conventional and well-known techniques. Forexample, the vector can be introduced directly into the host cells usingtechniques such as electroporation, microinjection, and biolisticmethods, such as particle bombardment.

Microinjection techniques are known in the art and well described in thescientific and patent literature. The introduction of DNA constructsusing polyethylene glycol precipitation is described, e.g., inPaszkowski et al. (1984). Electroporation techniques are described inFromm et al. (1985). Biolistic transformation techniques are describedin Klein et al. (1987).

For transforming plants or plant cells, the reporter constructs may alsobe combined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The virulencefunctions of the Agrobacterium tumefaciens host will direct theinsertion of the construct and adjacent marker into the plant cell DNAwhen the cell is infected by the bacteria. Agrobacteriumtumefaciens-mediated transformation techniques, including disarming anduse of binary vectors, are well described in the scientific literature.See, for example Horsch et al. (1984), and Fraley et al. (1983).

The host plant cells for screening reporter constructs can be from anyplant, including both dicots and monocots. Typically, plant cells arefrom Nicotiana benthamiana or Arabidopsis thaliana or another plant thatis routinely transformed and assayed in the art.

Other plants also can be used in the screening methods taught herein.These include cereals, for example, maize, sorghum, rice, wheat, barley,oats, rye, milo, flax, or gramma grass. Other plant genera include, butare not limited to, Cucurbita, Rosa, Vitis, Juglans, Gragaria, Lotus,Medicago, Onobrychis; Trigonella, Vigna, Citrus, Linum, Geranium,Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa,Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia,Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum,Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum,Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Allium, and Triticum.

Following transformation of the reporter constructs into the plant cell,the transformed cell or plant tissue is selected or screened byconventional techniques. The transformed cell or plant tissue containingthe reporter construct can then be regenerated, if desired, by knownprocedures. Additional methodology for the generation of plantscomprising expression constructs for screening chemicals can be found inthe art (see, e.g., U.S. Pat. No. 5,614,395).

Chemical Libraries

The compounds tested as modulators of yield regulators are typicallychemical compounds. Essentially any chemical compound of interest can beused to activate or down-regulate the activity of the promoters of theinstant description or to stabilize the polypeptides of the instantdescription using the assays as described. Most often, compounds can bedissolved in aqueous or organic (e.g., DMSO-based) solutions. The assaysare designed to screen large chemical libraries and usually includeautomating the assay steps, which are typically run in parallel (e.g.,in microtiter formats on microtiter plates in robotic assays). It willbe appreciated that there are many suppliers of chemical compounds,including Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-BiochemicaAnalytika (Buchs, Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical library containing a large number oftest compounds. Such “combinatorial chemical libraries” are thenscreened in one or more assays, as described herein, to identify thoselibrary members (particular chemical species or subclasses) thatactivate or down-regulate the activity of the promoters of the instantdescription. The compounds thus identified serve as conventional “leadcompounds” or can themselves be used as potential or actual agents fortreating plants or other organisms.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, small organic moleculelibraries (see, e.g., U.S. Pat. No. 5,569,588; thiazolidinones andmetathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos.5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337;and the like). Other chemistries for generating chemical diversitylibraries can also be used. Chemical diversity libraries are alsocommercially available, e.g., from such companies as 3-DimensionalPharmaceuticals Inc., Albany Molecular Research Inc., Alchemia Pty.Ltd., Argonaut Technologies Inc., ArQuie Inc, Biofocus DPI, ArrayBiopharma Inc., Axys Pharmaceutical Inc., Cambridge Combinatorial Ltd.,Charybdis Technologies Inc, ChemBridge Corp., CombiChem. Inc., ComGenexInc., Discovery Partners International Inc., Diversa Corp., EnzyMed.Inc. Versicor, Gryphon Sciences Inc, Ixsys Inc., Kosan Biosciences Inc.,Maxygen Inc., Molecumetics Ltd., Nanoscale Combinatorial Synthesis Inc.,Ontogen Corp., Orchid Biocompter Inc., Oxford Asymmetry Ltd., OxfordMolecular Group PLC, Panlabs Inc., Pharmacopeia Inc., Phytera Inc.,Proto Gene Inc., Sphere Biosystems Inc., Symyx Technologies Inc., andSystems Integration Drug Discovery Co.

Often, chemical libraries that are screened in the methods of theinstant description comprise compounds with molecular weights between150 and 600, an average c Log P value of 3 (range 0-9), an averagenumber of R-bonding acceptors of 3.5 (range 0-9), an average number ofR-bonding donors of one (range 0-4) and an average of three rotatablebonds (range 0-9). Such characteristics are typical of agrichemicalsknown in the art.

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries, are themselves commercially available(see, e.g., Chembridge, Inc., San Diego, Calif.; ComGenex, Princeton,N.J., Tripos, Inc., St. Louis, Mo., 3D Pharmaceuticals, Exton, Pa.,Martek Biosciences, Columbia, Md., etc.).

High Throughput Assays

In the high throughput assays, it is possible to screen up to severalthousand different test compounds in a single day. For example, eachwell of a microtiter plate can be used to run a separate assay against aselected test compound, or, if concentration or incubation time effectsare to be observed, every 5-10 wells can test a single test compound.Further, pools of test compounds can also be tested where 25 multiplecompounds are included in a single test sample. If a hit is thenidentified, the chemicals included in the pool can be individuallytested to identify an active compound.

The compounds selected from the reporter assays are also evaluated usingan additional screening step, for example, test compounds can be appliedto an organism of interest and evaluated by measuring the presence orabsence of a change in the level of the polypeptide of interest (if thecompound is selected from the translational fusion reporter system) or apolypeptide that is regulated by the target promoter (if the compound isselected from the transcriptional fusion reporter system) byconventional methods known in the art, for example, RT-PCR analysis,western blot analysis, microarray hybridization, or sequencing basedapproaches.

In some embodiments of the instant description, the test compoundsselected from the reporter gene system are subjected to a phenotypicanalysis. A phenotypic analysis involves treating an organism with thetest compound and detecting a modified trait which results from a changein the expression or activity of a polypeptide of interest, for example,a transcriptional regulatory polypeptide that regulates specificsignaling pathways. In some embodiments, phenotypic analyses wereperformed on plants, which typically involve assays of abiotic stresstolerance, such as water deprivation, dehydration, or osmotic stress, orassays that measure photosynthetic capacity.

Treatment of Plants

Once chemical compounds are identified and further validated inaccordance with the methods of the instant description, they can be usedto treat any plant, for example, vegetable, fruit, and orchard crops, toenhance plant performance.

Plants that can be treated include both monocots and dicots and inparticular, agriculturally important plant species, including but notlimited to, crops such as soybean, wheat, corn, potato, cotton, rice,oilseed rape (including canola), sunflower, alfalfa, sugarcane and turf;or fruits and vegetables, such as banana, blackberry, blueberry,strawberry, and raspberry, cantaloupe, carrot, cauliflower, coffee,cucumber, eggplant, grapes, honeydew, lettuce, mango, melon, onion,papaya, peas, peppers, pineapple, spinach, squash, sweet corn, tobacco,tomato, watermelon, rosaceous fruits (such as apple, peach, pear, cherryand plum) and vegetable brassicas (such as broccoli, cabbage,cauliflower, Brussels sprouts and kohlrabi). Other crops, fruits andvegetables whose phenotype may be changed include barley, currant,avocado, citrus fruits such as oranges, lemons, grapefruit andtangerines, artichoke, cherries, nuts such as the walnut and peanut,endive, leek, roots, such as arrowroot, beet, cassava, turnip, radish,yam, sweet potato and beans. Lower plants such as algae can also betreated in this manner.

The selected chemicals can be formulated for treating plants as a liquidor a solid form. For example, in liquid formulations, the plants can betreated with a spray, in a drench application, a drip application, orthrough irrigation. Formulations are prepared using known methodologyand may comprise other reagents conventionally employed in formulationof agricultural chemicals, e.g., emulsifying agents, surfactants, etc.Examples of formulations include emulsifiable concentrates, directlysprayable or dilutable solutions, dilute emulsions, wettable powders,soluble powders, dusts, granules or microcapsules. The methods ofapplication, such as spraying, atomizing, dusting, wetting, scatteringor pouring, are selected in accordance with the desired application. Forexample, a slow-release formulation can be applied as a soil treatmentso that a plant is exposed frequently to an isolated chemical (e.g.,turf grass). In other instance, it may be desirable to incorporate achemical compound selected in accordance with the method of the instantdescription into irrigation water for plants that experience frequentdroughts (e.g., cotton).

EXAMPLES Example 1 Transcriptional Fusion Reporter System

The transcriptional fusion reporter system of the present inventioncomprises, in operable linkage, at least: a reporter gene, a targetpromoter sequence recognized by a transcriptional regulatorypolypeptide, and a polynucleotide encoding a DNA sequence-specifictransactivator. The reporter gene expression is controlled by the targetpromoter sequence and the DNA sequence-specific transactivator.

One exemplar transcriptional fusion reporter system expresses adexamethasone-inducible transactivator, LEXA:GAL4:GR (SEQ ID NO: 1) andcomprises: a) a transcriptional regulatory polypeptide encoded by apolynucleotide that is operably linked to the opLEXA promoter that isresponsive to the dexamethasone-bound LEXA:GAL4:GR (SEQ ID NO: 2), andb) a reporter gene that is operably linked to the target promoter thatis recognized by the transcriptional regulatory polypeptide. A DBDsite::reporter1 component, for example, opLexA:reporter1, can also beincluded as a pre-characterization component to select cell lines andconstruct components that will impart strong inducible expression of thetranscriptional regulatory polypeptide; under such circumstances,candidate polynucleotide sequence comprising promoter and DBD:AD:GR, andpolynucleotide sequence of DBD site are introduced into a cell and theirsuitability for the screen are confirmed based on the ability ofactivating reporter expression (“reporter1”) in response todexamethasone treatment (FIG. 1).

Another exemplar transcriptional fusion reporter system comprises: a) apolynucleotide encoding a DNA sequence-specific transactivator LEXA:GAL4(SEQ ID NO: 4), which is operably linked to a target promoter that canbe recognized by a plant transcriptional regulatory polypeptide; b) areporter gene operably linked to a opLEXA promoter (SEQ ID NO: 5) (FIG.2). FIG. 9 shows an example of such a system, where a RD29A promoter isemployed to control the expression of a LEXA:GAL4 transactivator, which,in turn, activates the reporter gene expression through regulating theopLEXA promoter. This two component system has been shown to increasesignificantly the signal-to-noise ratio of the assay compared to thedirect fusion system, where the target promoter controls thetranscription of the reporter polynucleotide directly.

Another exemplar transcriptional fusion reporter system expresses adexamethasone-inducible DNA sequence-specific transactivator, e.g.,GAL4BD:VP16:GR (SEQ ID NO: 7). It also comprises: a) polynucleotide thatencodes a transcriptional regulatory polypeptide, operably linked to theGAL4UAS promoter region (SEQ ID NO: 11) that is responsive to thedexamethasone-bound GAL4BD:VP16:GR (SEQ ID NO: 7); b) a LEXA:GAL4polynucleotide (SEQ ID NO: 3) that is operably linked to a targetpromoter responsive to the transcriptional regulatory polypeptide; andc) a reporter gene that is operably linked to a opLEXA promoter (SEQ IDNO: 5) (FIG. 3). As described above, a DBD1 site::reporter1 componentcan also be included to select cell lines that can strongly induce theexpression of the target transcriptional regulatory polypeptide usingthe DBD1site promoter element.

These reporter constructs from the transcriptional fusion reportersystem can be introduced into plants or plant cells to screen forcompounds that can be used to modulate the activity of a target promoterthat is recognized by a transcriptional regulatory polypeptide. Thereporter gene expression can be monitored and compounds are selected onthe basis of their ability to alter reporter expression or activityrelative to controls.

Example 2 Translational Fusion Reporter System

The translational fusion reporter system of the present descriptioncomprises at least: 1) a polynucleotide encoding a DNA sequence specifictransactivator, 2) a polynucleotide encoding a fusion protein, and 3) anucleotide sequence recognized by the DNA sequence specifictransactivator. The genetic elements of 1), 2) and 3) are arranged in away such that the expression of a translational fusion of a reportermolecule and a polypeptide of interest is activated by the DNAsequence-specific transactivator.

One exemplar translational fusion reporter system expresses a LEXA:GAL4fusion (SEQ ID NO: 4) protein and comprises a polynucleotide encoding atranslational fusion of a reporter molecule and a polypeptide ofinterest that is operably linked to a opLEXA promoter (SEQ ID NO: 5)that is responsive to the LEXA:GAL4 (FIG. 4).

The reporter constructs of the translational fusion reporter system canbe introduced into plants or plant cells to screen for compounds thatcan modulate the stability of a polypeptide of interest. The reportergene expression can be monitored and compounds are selected on the basisof their ability to alter reporter expression or activity relative tocontrols. Hit compounds can be applied to plants and further validatedfor the ability to change the stability of the polypeptide of theinterest through biochemical analyses that are known in the art.

Example 3 The Two Component Transcription Fusion System has an ImprovedSignal-to-Noise Ratio Compared to a Direct Transcriptional Fusion

Aliquots of seeds for 10 independent lines harboring aprTARGET::LEXA:GAL4_opLEXA::GFP transgenic construct were surfacesterilized (see Example 9), where prTARGET comprises a promoter elementthat regulates a gene involved in desiccation stress tolerance ofArabidopsis, and distributed into a 96-well polystyrene plate at adensity of 5-10 seeds/well (n=16 wells) in standard liquid growthmedium. In parallel, seeds were sterilized and distributed for the topperforming prTARGET::GFP line previously identified from a study of over20 independent lines based on the largest fold-increase in GFP reporterlevels following an induction treatment which activates the promoterprTARGET. After six days of growth under 24 h hour light (100 microE m−2s−1) at 25 degrees C., eight wells/line were given the “inductiontreatment” and the other eight were “mock treated” and the plate wasreturned to the growth chamber. After 48 additional hours the plate wasremoved and fluorescence levels acquired using a Synergy HT multimodereader in area scan mode. FIG. 6 shows the average fluorescence and thestandard error of the mean. Several of the two-component linesout-performed the classic direct fusion using fold-induction andbackground fluorescence levels as the selection criteria.

Example 4 A Two-Component Transcriptional Fusion System Comprising aStress-Inducible Promoter RD29A

Seeds from 10 independent lines harboring aprRD29A::LEXA:GAL4_opLEXA::GFP were surface-sterilized and plated in thesame manner as described in Example 9 in parallel with thetop-performing prRD29A::GFP and prRD29B::GFP line, which were identifiedpreviously from a study of multiple independent lines based on thelargest fold-increase in GFP levels following an 1 μM ABA induction.Plants were grown in the same conditions as described in Example 3,except a 1 μM ABA induction which activates the RD29A and RD29Bpromoters was applied on day 5 of growth. Fluorescence levels ofindividual lines were acquired on day 8 and shown in FIG. 9. Several ofthe prRD29A::LEXA:GAL4_opLEXA::GFP exhibited significantly greater foldof GFP level increase upon ABA induction and less backgroundfluorescence levels compared to the direct fusions, e.g. prRD29A::GFPand prRD29B::GFP lines.

Example 5 Compound Screen Using the Two Component System

Seeds from Arabidopsis lines transformed with prTARGET::GFP (directfusion reporter system) or prTARGET::LexA:Gal4_oplexA::GFP (twocomponent reporter system) described in Example 3 were used in a primaryscreen on a 30K diverse compound panel to identify compounds that caninduce the prTARGET promoter using procedures described in Example 7.Compounds that showed at least 2.5 fold induction compared to DMSO wereidentified as “hits”. Several compounds that did not meet the thresholdof 2.5 fold induction with the direct fusion reporter line (Y axis) wereidentified as hits using the improved two-component system (X axis),demonstrating the improved sensitivity of the two-component system (FIG.7).

Twenty (20) compound hits, identified from the primary screen using theabove-referenced prTARGET::LexA:Gal4_oplexA::GFP reporter line, wereapplied to wild type Arabidopsis seedlings according to Examples 9-10 ina secondary screen. Desiccation tolerance assays were performedaccording to the methods describe in Example 13 below. Four of thesecompounds, “C71125”, “C66433”, “C71126” and “C71124”, were confirmed tohave conferred significant tolerance to desiccation stress toArabidopsis seedlings relative to the control, DMSO (FIG. 8).

Example 6 Identifying Compounds that Modulate Signaling PathwaysRelevant to Biotic Stress Tolerance, Abiotic Stress Tolerance, orNitrogen Usage

Biotic stresses or abiotic stresses can induce changes in geneexpression of signaling pathways that may potentiate a plant's naturaldefense against unfavorable conditions. A number of promoters have beenselected from some of the marker genes involved in these pathways, i.e.,genes that are up-regulated during the response of interest identifiedin the scientific literature or determined with in-house transcriptionalprofiling experiments. Promoters involved in signaling pathways ofinterest include but are not limited to drought-inducible promotersincluding sequences located in the promoter regions of At5g52310(RD29A), At5g52300, AT1G16850, At3g46230, AT1G52690, At2g37870,AT5G43840, At5g66780, At3g17520, and At4g09600, disease induciblepromoters including a regulatory sequence located in the promoter regionof AT1G15125; and those that are inducible by a change of nitrogenstatus in the environment including sequences located in the promoterregions of AT1G13300, AT2G48080, AT3G25790, AT5G10210, and AT5G19970.The exemplary promoter sequences that can be used to identify compoundsthat can modulate plant's drought response, disease response, ornitrogen usage include SEQ ID NO: 29-44.

Any one of the aforementioned promoter sequences or their functionalparts can be constructed into a two-component transcriptional fusionreporter system prGENE::LEXA:GAL4_opLEXA::GFP to screen for compoundsthat can induce the activity of prGENE and have beneficial effects onplant stress tolerance (prGENE represents any of the aforementionedpromoters). A primary screen using said reporter system is performed bythe method described in Example 7. Compounds that produce a greater thana pre-defined threshold level of induction of prGENE are identified as“hits” and applied to Arabidopsis wild-type seedlings according to theprocedures in Example 9 and 10. Phenotypic assays (used as secondaryscreens) are performed according to the methods in Example 13. Thecompounds that yield the desired phenotypes in plants, such as increasedstress tolerance, improved nitrogen use efficiency (NUE) and/or greaterdisease resistance, are then selected.

Example 7 Compound Screening

Sterile seeds are suspended in 0.5×MS, 0.5% sucrose, 0.05% MES and 0.1%Phytoblend agar at a density of 0.6 mg/mL and distributed to sterile96-well polystyrene plates (250 μL/well). The plates are covered andsealed with breathable tape and incubated at 25° C. under 24-hour light(100 μE m−2 s−1) in a germination growth chamber. After six days, theplates are removed and treated with the test compounds or DMSO (1 μL, induplicate plates), covered, sealed, and returned to the growth chamberfor one more day. The plates are then removed, uncovered and queued foranalysis. Green fluorescence protein (GFP, Ex 485 nm, Em 525 nm) isquantified on a TriStar Multimode Microplate Reader (Berthold,Rockville, Md.) in fluorescence area scan mode (3×3 grid, nine totalscans/well). The average fluorescence for the nine scans is divided bythe average fluorescence for all mock-treated wells (typically sixteenwells, 144 total scans) to obtain the per-plate activity ratio. The meanof the cross-plate duplicate activity ratios is then recorded.Additional statistical tests can be used to identify questionable sampledata. A one-tailed heteroscedastic t-test was used between the treatmentpopulation (nine data points) and the control population (144 datapoints) and the Benjamini-Hochberg adjustment for multiple testing togenerate a p(BH)-value was used for statistical significance. A highactivity ratio and poor p(BH)-value (>0.05) is typically a result of abiased distribution of fluorescence within the test well. This canresult from a seedling protruding towards the optical probe resulting inan erroneously high reading or punctate GFP expression from a dyingseedling due to compound toxicity.

Example 8 Screening of a Chemical Library Using a Screening Assay in aHigh Throughput Format

A transcriptional fusion reporter construct or a translation fusionreporter construct of the instant description is transformed intoplants. 1 μl each of the chemicals from a library purchased from acommercial source (such as ChemBridge™. Inc., San Diego, Calif.) isadded to 96 well plates containing in each well 5-10 Arabidopsis seeds,which harbor a reporter construct encoding GFP, for instance of the typeshown in FIGS. 1-4. The volume of the media in each well is 250 μl andthe final concentration of the chemical in each well is 28 μM. The seedsare allowed to germinate and grow in the medium. The data are normalizedbased on negative controls in the same plate that are not treated withthe chemical for one week and the GFP signal is quantified in a 96 wellfluorescent reader (TriStar, Berthold, Oak Ridge, Tenn.).

An alternative screening method involves the germination and growth ofthe Arabidopsis seedlings harboring the GFP construct in 96 well platesfor 4-7 days prior to the addition of the compound stock solutions. Theseedlings are exposed to the compound solution for an additional 1-3days and the GFP signal quantified in a 96 well fluorescent plate reader(TriStar, Berthold, Oak Ridge, Tenn.).

Example 9 Seed Preparation

Prior to plating, seeds for all experiments are surface sterilized inthe following manner: (1) 5 minute incubation with mixing in 70%ethanol; (2) 20 minute incubation with mixing in 30% bleach, 0.01%Triton® X-100; (3) five rinses with sterile water. Seeds are resuspendedin 0.1% sterile agarose and stratified at 4° C. for 2-4 days.

Example 10 Transplant Compound Treatment

Sterile stratified wild-type seeds (100 per plate) are sown on squareplates containing the following medium: 80% MS solution, 1% sucrose,0.05% MES, and 0.65% Phytoblend agar. Plates are incubated at 22° C.under 24-hour light (100 μE m−2 s−1) in a germination growth chamber. Onday 8, the seedlings are transferred to 6-well assay plates at a densityof 10 seedlings per well. The assay plates contained growth mediumspiked with a unique test compound or DMSO (carrier solvent, 0.4% v/v)per well. The compound-treated seedlings are incubated at 22° C. under24-hour light (100 μE m−2 s−1) in a germination growth chamber.

Example 11 Spray Compound Treatment Procedure

Sterile seeds (50 per plate) are sown on standard Petri dishescontaining the following medium: 80% MS solution, 1% sucrose, 0.05% MES,and 0.65% Phytagar. Plates are incubated at 22° C. under 24-hour light(95 μE m−2 s−1) in a germination growth chamber. On day 8, the seedlingsare transferred to square growth plates containing fresh medium (15-25seedlings per plate) and arranged such that their primary roots areexposed and aligned in parallel along the surface of the plate. Theplates are sealed with venting tape and returned to the growth chamber,oriented for vertical growth. Typically, on day 9, the plates aresprayed with a 0.01% Spreader Sticker surfactant solution containing thetest compound or DMSO (carrier solvent, 0.4% v/v) using a Preval®aerosol sprayer (1.5 mL/plate). The plates are re-sealed and returned tothe growth chamber (horizontal orientation). After an additional(assay-dependent) number of days in a growth chamber, the seedlings arethen subjected to any of the plate-based abiotic or biotic stressresistance assays detailed below. Alternatively, the plants may betreated by spraying on soil either once or multiple times during growthusing a formulated solution of the test compound (e.g. 0.01% SpreaderSticker); control plants are mock treated. The plants are then subjectedto phenotypic validation analysis by means of morphological,developmental or abiotic/biotic stress resistance assays, such as in theexample as described below.

Example 12 Genetic Marker Analysis

A compound identified in the screen analysis can also be evaluated forthe effects on the genetic markers of a signaling pathway underphysiological conditions where this signaling pathway is active. Suchgenetic marker assays are typically conducted after or concurrently withthe primary screen measuring the activation or repression of thepromoter-reporter construct, and can be performed using a variety oftechniques known by those of skill in the art to be suitable. Forexample, mRNA expression can be detected using amplification-basedmethodologies, northern or dot blots, nuclease protection and the like.Polypeptide products can be identified using immunoassays. For example,to evaluate a compound for its effect on the genetic components(promoters or polypeptides) of the light signaling pathway, Arabidopsisseedlings are grown on solid media (50% MSIB5, 0.05% MES (PH 5.7), 0.5%sucrose, 0.8% agar) in a growth chamber at 22° C. with continuous light(95IlMollm2/s) for nine days. The seedlings are then transplanted ontomedia containing various chemicals (typically at 20 μM) or DMSO controlsand returned to identical growth conditions for 6 h or 24 h. At theindicated time, the seedlings are removed from the media and immediatelyfrozen in liquid nitrogen. RNA is extracted and cDNA is prepared usingstandard procedures known in the art. RT-PCR analysis is performed usingprimers for the genes that are critical component of the light signalingpathway, such as, for example, SEQ ID NOs: 2 and 28 of PCT publicationWO2009/117448, the entire content of which is incorporated herein byreference.

Genetic marker activation or inhibition can also be determined by usingreporter constructs. Such reporter constructs can, e.g., comprise thepromoter sequences from the genetic markers, or alternatively, cancomprise promoters form genes that are responsive to the geneticmarkers. Activation or inhibition using reporter constructs can beanalyzed using the same methodology as that employed for evaluating thepromoter-reporter activation/inhibition.

Example 13 Phenotypic Validation Analysis

In these Examples, unless otherwise indicated, morphological andphysiological traits are disclosed for plants that are treated by a testcompound in comparison to those treated by a control compound or acarrier solvent under the identical environmental conditions. Thus, aplant treated with a test compound that is described as large and/ordrought tolerant is large and more tolerant to drought with respect to acontrol plant, the latter including plants treated with a controlcompound or a carrier solvent or no treatment. When a plant is said tohave a better performance than controls, it generally is larger, havegreater yield, and/or show less stress symptoms than control plants. Thebetter performing lines may, for example, have produced lessanthocyanin, or are larger, greener, more turgid, or more vigorous whenchallenged with a particular stress, compared to controls as notedbelow. Better performance generally implies greater size or yield, ortolerance to a particular biotic or abiotic stress, less sensitivity toABA, or better recovery from a stress (as in the case of a soil-baseddrought treatment) than controls.

Phenotypic analyses can be performed according to what is known in theart, or with the following methods.

Morphological Analysis

Morphological analysis is performed to determine whether changes intranscriptional regulatory polypeptide levels or compound treatmentaffect plant growth and development. Arabidopsis seeds are cold-treated(stratified) on plates for three days in the dark (in order to increasegermination efficiency) prior to transfer to growth cabinets. Initially,plates are incubated at 22° C. under a light intensity of approximately100 microEinsteins for seven days. Seedlings (treated or untreated asdescribed in Example 10 or 11) are then transferred onto soil (Sunshine®potting mix) Following transfer to soil, trays of seedlings are coveredwith plastic lids for 2-3 days to maintain humidity while they becomeestablished. Plants are grown on soil under fluorescent light at anintensity of 70-95 microEinsteins at a temperature of 18-23° C. and areoptionally subjected to chemical treatments (or mock treatments) asdescribed in Example 10 or 11. Light conditions consist of a 24-hourphotoperiod unless otherwise stated. In instances where alterations inflowering time are apparent, flowering time may be re-examined under8-hour, 12-hour and 24-hour light to assess whether the phenotype isphotoperiod dependent. Under typical 24-hour light growth conditions,the typical generation time (seed to seed) for Arabidopsis isapproximately 14 weeks.

Because many aspects of Arabidopsis development are dependent onlocalized environmental conditions, in all cases plants are evaluated incomparison to controls (i.e. plants that are untreated or treated with acontrol compound or a solvent carrier and are otherwise identical to theplants treated with the test compounds) in the same flat. Carefulexamination is made at the following stages: seedling (1 week), rosette(2-3 weeks), flowering (4-7 weeks), and late seed set (8-12 weeks). Seedis also inspected. Plants having no or few seeds are consideredpartially or totally sterile. Seedling morphology is assessed onselection plates. At all other stages, plants are macroscopicallyevaluated while growing on soil or another suitable growth medium. Allsignificant differences (including alterations in growth rate, size,leaf and flower morphology, coloration and flowering time) are recorded,but routine measurements are not be taken if no differences areapparent. In certain cases, stem sections are stained to reveal lignindistribution. In these instances, hand-sectioned stems are mounted inphloroglucinol saturated 2M HCl (which stains lignin pink) and viewedimmediately under a dissection microscope.

Physiological Analysis

Ten lines are typically examined in subsequent plate based physiologyassays. A similar number of compound-treated plants are compared tocontrols when testing the effects of compound treatments.

Nitrogen Use Efficiency (NUE) Assay

There are multiple ways in which a plant can change nitrogenpartitioning in response to changes in nitrogen availability. Forexample increased photosynthesis/seed dry weight or biomass. Routinemeasurements can be used to identify beneficial changes in nitrogenpartitioning that result in plants with improved NUE.

One or multiple of the following parameters are assessed to determinethe benefit to compound treated plants versus mock-treated controllines:

Photosynthesis: Light Saturated/Light Limited/Vcmax/Jmax/TPU limitation

Electron Transport: Light Saturated/Light limited

Respiration: Whole plant

Leaf chlorophyll content

Plant dry weight (root/shoot)

Plant carbon:nitrogen ratios (root/shoot)

Seed dry weight

Compound treatments that cause plant samples to deviate from controls inany of these relationships may improve nitrogen usage.

Plate Assays

Different plate-based physiological assays (shown below), representing avariety of abiotic and water-deprivation-stress related conditions, areused as a pre-screen to identify top performing lines (i.e. linestreated with a particular compound), that are generally then tested insubsequent soil based assays. Typically, ten lines are subjected toplate assays, from which the best three lines are selected forsubsequent soil based assays.

In addition, a nutrient limitation assay can be used to find compoundsthat allow more plant growth upon deprivation of nitrogen. Nitrogen is amajor nutrient affecting plant growth and development that ultimatelyimpacts yield and stress tolerance. These assays monitor primarily rootbut also rosette growth on nitrogen deficient media. In all higherplants, inorganic nitrogen is first assimilated into glutamate,glutamine, aspartate and asparagine, the four amino acids used totransport assimilated nitrogen from sources (e.g. leaves) to sinks (e.g.developing seeds). This process may be regulated by light, as well as byC/N metabolic status of the plant. A C/N sensing assay is thus used tolook for alterations in the mechanisms plants use to sense internallevels of carbon and nitrogen metabolites which could activate signaltransduction cascades that regulate the transcription of N-assimilatorygenes. To determine whether these mechanisms are altered or modified, weexploit the observation that control plants grown on media containinghigh levels of sucrose (3%) without a nitrogen source accumulate highlevels of anthocyanins. This sucrose-induced anthocyanin accumulationcan be relieved by the addition of either inorganic or organic nitrogen.Glutamine is used as a nitrogen source since it also serves as acompound used to transport N in plants.

Growth Assays

Unless otherwise stated, experiments are typically performed with theArabidopsis thaliana ecotype Columbia (col-0), soybean or maize plants.

Growth assays may be conducted with Arabidopsis or other plant species(e.g., soy, maize, etc.) that are treated or untreated with testcompounds or control as described in Examples 10 or 11. For example,Arabidopsis seedlings are grown on solid media (50% MS/B5, 0.05% MES (pH5.7), 0.5% sucrose, 0.8% agar) in a growth chamber at 22° C. withcontinuous light (95 μMol/m2/s) for nine days. The seedlings are thentransplanted onto media containing various chemicals (typically at 20μM) or DMSO controls and returned to identical growth conditions forthree additional days. Growth assays may assess tolerance to severedesiccation (a type of water deprivation assay), growth in coldconditions at 8° C., root development (visual assessment of lateral andprimary roots, root hairs and overall growth), and phosphate limitation.

For the nitrogen limitation assay, plants are grown in 80% Murashige andSkoog (MS) medium in which the nitrogen source is reduced to 20 mg/L ofNH₄NO₃. Note that 80% MS normally has 1.32 g/L NH₄NO₃ and 1.52 g/L KNO₃.

For phosphate limitation assays, seven day old seedlings are germinatedon phosphate-free MS medium in which KH₂PO₄ is replaced by K₂SO₄.

For chilling growth assays, seeds are germinated and grown for sevendays on MS+Vitamins+1% sucrose at 22° C. and are then transferred tochilling conditions at 8° C. and evaluated after another 10 days and 17days.

For desiccation (plate-based water deprivation) assays, sterile,stratified wild-type seeds (100 per plate) were sown on square platesand on day 8 the seedlings were subjected to treatment by a unique testcompound or a control compound according to Example 10 or 11. On day 11the assay plates were photographed and placed in a laminar flow hoodwith the lid removed for 3 hours, rotating the plates 180 degrees after90 minutes. The seedlings were then removed from the medium, placed onthe surface of the inverted lid and desiccated an additional 3.6 hours.The seedlings were then transferred to square plates containing freshgrowth medium, returned to the growth chamber and allowed to recover for3-4 days prior to photo documentation and scoring.

For the polyethylene glycol (PEG) hyperosmotic stress tolerance screen,plant seeds are gas sterilized with chlorine gas for 2 h. The seeds areplated on each plate containing 3% PEG, ½×MS salts, 1% phytagel, and 10μg/ml glufosinate-ammonium (BASTA). Two replicate plates per seed lineare planted. The plates are placed at 4° C. for three days to stratifyseeds. The plates are held vertically for 11 additional days attemperatures of 22° C. (day) and 20° C. (night). The photoperiod is 16h. with an average light intensity of about 120 μmol/m2/s. The racksholding the plates are rotated daily within the shelves of the growthchamber carts. At 11 days, root length measurements are made. At 14days, seedling status is determined, root length is measured, growthstage is recorded, the visual color is assessed, pooled seedling freshweight is measured, and a whole plate photograph is taken.

Germination assays may also be carried out with NaCl (150 mM, to measuretolerance to salt), sucrose (9.4%, to measure altered or modified sugarsensing), cold (8° C.) or heat (32° C.). All germination assays areperformed in aseptic conditions. Growing the plants under controlledtemperature and humidity on sterile medium produces uniform plantmaterial that has not been exposed to additional stresses (such as waterstress) which could cause variability in the results obtained.

Prior to plating, seed for all experiments are surface sterilized in thefollowing manner: (1)₅ minute incubation with mixing in 70% ethanol, (2)20 minute incubation with mixing in 30% bleach, 0.01% triton-X 100, (3)5× rinses with sterile water, (4) Seeds are re-suspended in 0.1% sterileagarose and stratified at 4° C. for 3-4 days. All germination assaysfollow modifications of the same basic protocol. Sterile seeds may besown on conditional media that has a basal composition of 80%MS+Vitamins, or media containing test compounds as described in Example6 above. Plates may be incubated at 22° C. under 24-hour light (120-130μE m−2 s−1) in a growth chamber. Evaluation of germination and seedlingvigor may be performed five days after planting.

Chlorophyll content, an indicator of photosynthetic capacity, may bemeasured with a SPAD meter.

Wilt Screen Assay

Soybean plants treated with test compounds or DMSO are grown in 5″ potsin growth chambers. After the seedlings reach the V1 stage (the V1 stageoccurs when the plants have one trifoliolate, and the unifoliolate andfirst trifoliolate leaves are unrolled), water is withheld and thedrought treatment thus started. A drought injury phenotype score isrecorded, in increasing severity of effect, as 1 to 4, with 1 designatedno obvious effect and 4 indicating a dead plant. Drought scoring isinitiated as soon as one plant in one growth chamber had a drought scoreof 1.5. Scoring continues every day until at least 90% of the wild typeplants achieve scores of 3.5 or more. At the end of the experiment thescores for both test compound treated and control soybean seedlings arestatistically analyzed using Risk Score and Survival analysis methods(Glantz (2001); Hosmer and Lemeshow (1999).

Water Use Efficiency (WUE) Assay

WUE is estimated by exploiting the observation that elements can existin both stable and unstable (radioactive) forms. Most elements ofbiological interest (including C, H, O, N, and S) have two or morestable isotopes, with the lightest of these being present in muchgreater abundance than 14_(C)=the others. For example, ¹²C is moreabundant than ¹³C in nature (¹²C=98.89%, ¹³C=1.11%, ¹⁴C=<10-10%).Because ¹³C is slightly larger than ¹²C, fractionation of CO₂ duringphotosynthesis occurs at two steps:

1. ¹²CO₂ diffuses through air and into the leaf more easily;

2. ¹²CO₂ is preferred by the enzyme in the first step of photosynthesis,ribulose bisphosphate carboxylase/oxygenase.

WUE has been shown to be negatively correlated with carbon isotopediscrimination during photosynthesis in several C3 crop species. Carbonisotope discrimination has also been linked to drought tolerance andyield stability in drought-prone environments and has been successfullyused to identify genotypes with better drought tolerance. ¹³C/¹²Ccontent is measured after combustion of plant material and conversion toCO₂, and analysis by mass spectroscopy. With comparison to a knownstandard, ¹³C content is altered in such a way as to suggest thattreatment with test compounds improves water use efficiency.

Another potential indicator of WUE is stomatal conductance, that is, theextent to which stomata are open.

Data Interpretation

At the time of evaluation, plants are typically given one of thefollowing qualitative scores:

-   -   (++) Substantially enhanced performance compared to controls.        The phenotype is very consistent and growth is significantly        above the normal levels of variability observed for that assay.    -   (+) Enhanced performance compared to controls. The response is        consistent but is only moderately above the normal levels of        variability observed for that assay.    -   (wt) No detectable difference from wild-type controls.    -   (−) Impaired performance compared to controls. The response is        consistent but is only moderately above the normal levels of        variability observed for that assay.    -   (−−) Substantially impaired performance compared to controls.        The phenotype is consistent and growth is significantly above        the normal levels of variability observed for that assay.

(n/d) Experiment failed, data not obtained, or assay not performed.

Soil Drought (Clay Pot)

The soil drought assay (typically performed on Arabidopsis in clay pots)is based on that described by Haake et al. (2002).

Sterile seeds (50 per plate) are sown on standard Petri dishescontaining the following medium: 80% MS solution, 1% sucrose, 0.05% MES,and 0.65% Phytagar. Plates are incubated at 22° C. under 24-hour light(95 μE m−2 s−1) in a germination growth chamber. After seven days ofgrowth the seedlings are transplanted to 3.5 inch diameter clay potscontaining 80 g of a 50:50 mix of vermiculite:perlite topped with 80 gof ProMix. Typically, each pot contains 14 evenly spaced seedlings. Thepots are maintained in a growth room under 24-hour light conditions(18-23° C., and 90-100 μE m−2 s−1) and watered for a period of 14 days.Compounds (or DMSO) are applied as a 0.01% Spreader Sticker solution (orsimilar formulation) using a Preval aerosol sprayer (ca. 2 mL/pot or 100g/ha) no more than three times during days 7-13 post-transplant. Wateris then withheld and pots are placed on absorbent diaper paper for aperiod of 8-10 days to apply a drought treatment. At the end of thedrought period, pots are re-watered and then scored after 5-6 additionaldays. The number of surviving plants in each pot is counted, and thesurvival percentage calculated.

In a given experiment, six or more pots of plants treated by testcompounds with six or more pots of the appropriate control are typicallycompared. The mean drought score and mean proportion of plants surviving(survival rate) are calculated for both the transgenic line and thewild-type pots. In each case a p-value* is calculated, which indicatesthe significance of the difference between the two mean values.

For the assays where control and experimental plants are in separatepots, survival is analyzed with a logistic regression to account for thefact that the random variable is a proportion between 0 and 1. Thereported p-value is the significance of the experimental proportioncontrasted to the control, based upon regressing the logit-transformeddata.

Drought score, being an ordered factor with no real numeric meaning, isanalyzed with a non-parametric test between the experimental and controlgroups. The p-value is calculated with a Mann-Whitney rank-sum test.

Disease Resistance

Resistance to pathogens, such as Sclerotinia sclerotiorum and Botrytiscinerea, can be assessed in plate-based assays. Unless otherwise stated,all experiments are performed with the Arabidopsis thaliana ecotypeColumbia (Col-0). Control plants for assays on lines containing directpromoter-fusion constructs are wild-type plants or Col-0 plantstransformed an empty transformation vector (pMEN65).

Prior to plating, seed for all experiments are surface sterilized in thefollowing manner: (1) 5 minute incubation with mixing in 70% ethanol;(2) 20 minute incubation with mixing in 30% bleach, 0.01% Triton X-100™;(3) five rinses with sterile water. Seeds are resuspended in 0.1%sterile agarose and stratified at 4° C. for 2-4 days.

Sterile seeds are sown on starter plates (15 mm deep) containing 50% MSsolution, 1% sucrose, 0.05% MES, and 1% Bacto™-Agar. 40 to 50 seeds aresown on each plate. Seedlings are grown on solid media (50% MS/B5, 0.05%MES (pH 5.7), 0.5% sucrose, 0.8% agar) in a growth chamber at 22° C.with continuous light (95 μMol/m2/s) for nine days. The seedlings arethen transplanted onto media containing various chemicals (typically at20 μM) or DMSO controls and returned to identical growth conditions forthree additional days. Seedlings are then transferred to assay plates(25 mm deep plates with medium minus sucrose). On day 14, seedlings areinoculated (specific method below). After inoculation, plates are put ina growth chamber under a 12-hour light/12-hour dark schedule. Lightintensity is lowered to 70-80 μE m−2 s−1 for the disease assay.

Sclerotinia inoculum preparation. A Sclerotinia liquid culture isstarted three days prior to plant inoculation by cutting a small agarplug (¼ sq. inch) from a 14- to 21-day old Sclerotinia plate (on PotatoDextrose Agar; PDA) and placing it into 100 ml of half-strength PotatoDextrose Broth. The culture is allowed to grown in the Potato DextroseBroth at room temperature under 24-hour light for three days. On the dayof seedling inoculation, the hyphal ball is retrieved from the medium,weighed, and ground in a blender with water (50 ml/gm tissue). Aftergrinding, the mycelial suspension is filtered through two layers ofcheesecloth and the resulting suspension is diluted 1:5 in water. Plantsare inoculated by spraying to run-off with the mycelial suspension usinga Preval aerosol sprayer.

Botrytis inoculum preparation. Botrytis inoculum is prepared on the dayof inoculation. Spores from a 14- to 21-day old plate (on PDA) areresuspended in a solution of 0.05% glucose, 0.03M KH₂PO₄ to a finalconcentration of 10⁴ spores/ml. Seedlings are inoculated with a Prevalaerosol sprayer, as with Sclerotinia inoculation.

Resistance to Erysiphe cichoracearum is assessed in a soil-based assay.Erysiphe cichoracearum is propagated on a pad4 mutant line in the Col-0background, which is highly susceptible to Erysiphe (Reuber et al.(1998), or on squash plants, since this particular species of Erysiphealso parasitizes squash. Inocula are maintained by using a smallpaintbrush to dust conidia from a 2-3 week old culture onto 4-week oldplants. For the assay, seedlings are grown on plates for one week under24-hour light in a germination chamber, then transplanted to soil andgrown in a walk-in growth chamber under a 12-hour light/12-hour darklight regimen, 70% humidity. Each line is transplanted to two 13 cmsquare pots, nine plants per pot. In addition, three control plants aretransplanted to each pot, for direct comparison with the test line.Approximately 3.5 weeks after transplanting, plants are inoculated usingsettling towers, as described by Reuber et al., 1998. Generally, threeto four heavily infested leaves are used per pot for the disease assay.Level of fungal growth is evaluated eight to ten days after inoculation.

It is expected that the same methods may be applied to identify otheruseful and valuable promoter sequences, and the sequences may be derivedfrom a diverse range of species.

Embodiments of the Instant Description Embodiment 1

One or more reporter gene constructs comprising (a) a target promotersequence that is capable of being recognized by a transcription factor,(b) a polynucleotide sequence that encodes a DNA sequence-specifictransactivator, and (c) a reporter polynucleotide,

wherein the target promoter sequence and the DNA sequence-specifictransactivator cooperatively regulate expression of the reporterpolynucleotide.

Embodiment 2

The one or more reporter gene constructs of Embodiment 1, wherein thesequence specific transactivator is capable of binding a regulatoryregion of the reporter polynucleotide.

Embodiment 3

The one or more reporter gene constructs of Embodiment 1, wherein thesequence specific transactivator is capable of binding a regulatoryregion of a polynucleotide that encodes the transcription factor.

Embodiment 4

The one or more reporter gene constructs of Embodiment 1, wherein thetranscription factor is capable of binding a regulatory region of thereporter polynucleotide.

Embodiment 5

The one or more reporter gene constructs of Embodiment 1, wherein theDNA sequence-specific transactivator is DBD:AD;

wherein AD comprised at least one sequence selected from the groupconsisting of SEQ ID NOs: 20, and 22-26; and

wherein DBD comprised at least one sequence selected form the groupconsisting of SEQ ID NOs: 16 and 18.

Embodiment 6

The one or more reporter gene constructs of Embodiment 1, wherein theDNA sequence-specific transactivator is a steroid-inducibletransactivator, which regulates transcription of the transcriptionfactor when bound by a steroid.

Embodiment 7

The one or more reporter gene constructs of Embodiment 6, wherein thesteroid-inducible transactivator is DBD:AD:GR;

wherein AD comprises at least one sequence selected from the groupconsisting of SEQ ID NOs: 20, and 22-26;

wherein DBD comprises at least one sequence selected from the groupconsisting of SEQ ID NOs: 16 and 18;

wherein GR comprises SEQ ID NO: 14; and

wherein the steroid is dexamethasone.

Embodiment 8

The one or more reporter gene constructs of Embodiment 6, wherein theone or more reporter gene constructs further comprise a polynucleotidesequence that encodes an additional DNA sequence-specifictransactivator,

wherein the additional DNA sequence-specific transactivator regulatestranscription of the reporter gene through binding of its cognatesequence that is operably linked to the reporter polynucleotide.

Embodiment 9

The one or more reporter gene constructs of Embodiment 8, wherein thetranscription factor is capable of binding a regulatory region of apolynucleotide encoding the additional DNA sequence-specifictransactivator.

Embodiment 10

The one or more reporter gene constructs of Embodiment 8, wherein thesteroid-inducible transactivator is DBD:AD:GR;

wherein AD is selected from the group consisting of SEQ ID NOs: 20, and22-26;

wherein DBD is selected form the group consisting of SEQ ID NOs: 16 and18;

wherein GR comprises SEQ ID NO: 14; and

wherein the steroid is dexamethasone.

Embodiment 11

One or more reporter gene constructs comprising a polynucleotideencoding a DNA sequence-specific transactivator and a polynucleotideencoding a translational fusion of a reporter gene molecule and apolypeptide of interest,

wherein the DNA sequence-specific transactivator regulates expression ofthe translational fusion.

Embodiment 12

The one or more reporter gene constructs of Embodiment 11, wherein theDNA sequence-specific transactivator is DBD:AD:GR;

wherein AD is selected from the group consisting of SEQ ID NOs: 20, and22-26;

wherein DBD is selected form the group consisting of SEQ ID NOs: 16 and18; and

wherein GR comprises SEQ ID NO: 14.

Embodiment 13

A transgenic cell comprising (a) a target promoter sequence that iscapable of being recognized by a transcription factor, (b) apolynucleotide sequence that encodes a DNA sequence-specifictransactivator, and (c) a reporter polynucleotide,

wherein the target promoter and the DNA sequence-specific transactivatorcooperatively regulates expression of the reporter gene.

Embodiment 14

The transgenic cell of Embodiment 13, wherein the sequence specifictransactivator is capable of binding a regulatory region of the reporterpolynucleotide.

Embodiment 15

The transgenic cell of Embodiment 13, wherein the sequence specifictransactivator is capable of binding a regulatory region of apolynucleotide that encodes the transcription factor.

Embodiment 16

The transgenic cell of Embodiment 13, wherein the transcription factoris capable of binding a regulatory region of the reporterpolynucleotide.

Embodiment 17

The transgenic cell of Embodiment 13, wherein the DNA sequence-specifictransactivator is DBD:AD:GR;

wherein AD comprises at least one sequence selected from the groupconsisting of SEQ ID NOs: 20, and 22-26;

wherein DBD comprises at least one sequence selected form the groupconsisting of SEQ ID NOs: 16 and 18; and

wherein GR comprises SEQ ID NO: 14.

Embodiment 18

The transgenic cell of Embodiment 13, wherein the DNA sequence-specifictransactivator is a steroid-inducible transactivator, which regulatestranscription of the transcription factor when bound by a steroid.

Embodiment 19

The transgenic cell of Embodiment 18, wherein the steroid-inducibletransactivator is

DBD:AD:GR;

wherein AD comprises at least one sequence selected from the groupconsisting of SEQ ID NOs: 20, and 22-26;

wherein DBD comprises at least one sequence selected form the groupconsisting of SEQ ID NOs: 16 and 18;

wherein GR comprises SEQ ID NO: 14; and

wherein the steroid is dexamethasone.

Embodiment 20

The transgenic cell of Embodiment 18, further comprises a polynucleotidesequence that encodes an additional DNA sequence-specific transactivatorand is located at 3′ relative to the steroid-inducible transactivatorand 5′ relative to the reporter gene,

wherein the additional DNA-sequence-specific transactivator regulatestranscription of the reporter gene through binding of its cognatesequence that is operably linked to the reporter polynucleotide.

Embodiment 21

The transgenic cell of Embodiment 19, wherein the transcription factoris capable of binding a regulatory region of a polynucleotide encodingthe additional DNA sequence-specific transactivator.

Embodiment 22

The transgenic cell of Embodiment 19, wherein the steroid-inducibletransactivator is

DBD:AD:GR;

wherein AD comprises at least one sequence selected from the groupconsisting of SEQ ID NOs: 20, and 22-26;

wherein DBD comprises at least one sequence selected form the groupconsisting of SEQ ID NOs: 16 and 18;

wherein GR comprises SEQ ID NO: 14; and

wherein the steroid is dexamethasone.

Embodiment 23

A transgenic cell comprising a polynucleotide encoding a DNAsequence-specific transactivator and a polynucleotide encoding atranslational fusion of a reporter gene molecule and a polypeptide ofinterest,

wherein the DNA sequence-specific transactivator regulates expression ofthe translational fusion.

Embodiment 24

The transgenic cell of Embodiment 23, wherein the DNA sequence-specifictransactivator is DBD:AD:GR;

wherein AD comprises at least one sequence selected from the groupconsisting of SEQ ID NOs: 20, and 22-26;

wherein DBD comprises at least one sequence selected from the groupconsisting of SEQ ID NOs: 16 and 18; and

wherein GR comprises SEQ ID NO: 14.

Embodiment 25

The transgenic cell of Embodiment 13, wherein the transgenic cell is acell derived from plants, mammals, microbes, Drosophila, Caenorhabditis,or yeast.

Embodiment 26

A method of screening for useful compounds comprising the steps of:

-   -   (a) contacting at least one test compound with a transgenic cell        comprising a target promoter sequence that is capable of being        recognized by a transcription factor, a polynucleotide sequence        that encodes a DNA sequence-specific transactivator, and a        reporter polynucleotide,        -   wherein the target promoter and the DNA sequence-specific            transactivator cooperatively regulate expression of the            reporter gene; and    -   (b) selecting a compound that alters the reporter gene activity        relative to controls.

Embodiment 27

The method of Embodiment 26, wherein the sequence specifictransactivator is capable of binding a regulatory region of the reporterpolynucleotide.

Embodiment 28

The method of Embodiment 26, wherein the sequence specifictransactivator is capable of binding a regulatory region of apolynucleotide that encodes the transcription factor.

Embodiment 29

The method of Embodiment 26, wherein the transcription factor is capableof binding a regulatory region of the reporter polynucleotide.

Embodiment 30

The method of Embodiment 26, wherein the DNA sequence-specifictransactivator is

DBD:AD:GR;

wherein AD comprises at least one sequence selected from the groupconsisting of SEQ ID NOs: 20, and 22-26;

wherein DBD comprises at least one sequence selected form the groupconsisting of SEQ ID NOs: 16 and 18; and

wherein GR comprises SEQ ID NO: 14.

Embodiment 31

The method of Embodiment 26, wherein the DNA sequence-specifictransactivator is a steroid-inducible transactivator, which regulatestranscription of the transcription factor when bound by a steroid.

Embodiment 32

The method of Embodiment 31, wherein the steroid-inducibletransactivator is

DBD:AD:GR;

wherein AD comprises at least one sequence selected from the groupconsisting of SEQ ID NOs: 20, and 22-26;

wherein DBD comprises at least one sequence selected form the groupconsisting of SEQ ID NOs: 16 and 18;

wherein GR comprises SEQ ID NO: 14; and

wherein the steroid is dexamethasone.

Embodiment 33

The method of Embodiment 31, wherein the transgenic cell furthercomprises a polynucleotide sequence that encodes an additional DNAsequence-specific transactivator,

wherein the additional DNA-sequence-specific transactivator regulatestranscription of the reporter gene through binding of its cognatesequence that is operably linked to the reporter polynucleotide.

Embodiment 34

The method of Embodiment 33, wherein the transcription factor is capableof binding a regulatory region of a polynucleotide encoding theadditional DNA sequence-specific transactivator.

Embodiment 35

The method of Embodiment 33, wherein the dexamethasone steroid-inducibletransactivator is

DBD:AD:GR;

wherein AD comprises at least one sequence selected from the groupconsisting of SEQ ID NOs: 20, and 22-26;

wherein DBD comprises at least one sequence selected form the groupconsisting of SEQ ID NOs: 16 and 18;

wherein GR comprises SEQ ID NO: 14; and

wherein the steroid is dexamethasone.

Embodiment 36

A method of screening for useful compounds comprising the steps of:

(a) contacting at least one test compound with a transgenic cellcomprising a polynucleotide encoding a DNA sequence-specifictransactivator and a polynucleotide encoding a translational fusion of areporter gene molecule and a polypeptide of interest,

-   -   wherein the DNA sequence-specific transactivator regulates        expression of the translational fusion; and

(b) selecting a compound that alters the reporter gene activity relativeto controls.

Embodiment 37

The method of Embodiment 36, wherein the DNA sequence-specifictransactivator is

DBD:AD:GR;

wherein AD comprises at least one sequence selected from the groupconsisting of SEQ ID NOs: 20, and 22-26;

wherein DBD comprises at least one sequence selected form the groupconsisting of SEQ ID NOs: 16 and 18; and

wherein GR comprises SEQ ID NO: 14.

Embodiment 38

The method of Embodiment 26 further comprising the step of:

-   -   (c) contacting a plant with the selected compound and detecting        a modified trait in the plant relative to controls.

Embodiment 39

The method of Embodiment 36 further comprising the step of:

-   -   (c) contacting a plant with the selected compound and detecting        a modified trait in the plant relative to controls.

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All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The present description is not limited by the specific embodimentsdescribed herein. The instant description now being fully described, itwill be apparent to one of ordinary skill in the art that many changesand modifications can be made thereto without departing from the spiritor scope of the appended claims. Modifications that become apparent fromthe foregoing description and accompanying figures fall within the scopeof the claims.

What is claimed is:
 1. A method of screening for a hit compound thatincreases plant desiccation tolerance, performance or yield, said methodcomprising the steps of: (a) contacting at least one test compound witha transgenic plant cell comprising: (i) a target drought-induciblepromoter sequence that is operably linked to a polynucleotide sequencethat encodes a DNA sequence-specific transactivator; and (ii) a reporterpolynucleotide that is operably linked to a promoter sequence that canbe recognized by the DNA sequence-specific transactivator; wherein thetarget drought-inducible promoter sequence can be recognized by atranscriptional regulatory polypeptide that modulates a signalingpathway and said modulation results in increased tolerance todesiccation, increased plant performance or increased yield in a plant;(b) identifying a test compound that alters the reporter gene activityrelative to controls and selecting a hit compound so identified; and (c)in a secondary screen, when said hit compound is applied to the plantsaid hit compound increases the tolerance to desiccation, plantperformance, or yield of the plant.
 2. The method of claim 1, whereinthe DNA sequence-specific transactivator is a translational fusion of aDNA binding domain (DBD) and a transcriptional activation domain (AD);wherein the AD comprises SEQ ID NO: 20; and wherein the DBD comprisesSEQ ID NO:
 16. 3. The method of claim 1, wherein the reporterpolynucleotide encodes green fluorescent protein.
 4. The method of claim1, wherein the target promoter sequence is an At3g46230 promoter or afunctional part thereof having a promoter function.
 5. The method ofclaim 1, wherein the transgenic cell further comprises a polynucleotidesequence that encodes an additional DNA sequence-specifictransactivator, wherein transcription of the polynucleotide sequenceencoding the additional DNA-sequence-specific transactivator is underthe control of a promoter sequence that is recognized by the firstDNA-sequence-specific transactivator; and wherein the additionalDNA-sequence-specific transactivator regulates transcription of thereporter gene through binding of its cognate sequence that is operablylinked to the reporter polynucleotide.
 6. The method of claim 1 furthercomprising the step of: (c) contacting a plant with the hit compound anddetecting the increased tolerance to desiccation, performance, or yieldin the plant relative to controls.
 7. A method of screening for acompound that increases tolerance to desiccation of a plant, said methodcomprising the steps of: (a) contacting at least one test compound witha transgenic plant cell comprising: (i) a target drought-induciblepromoter sequence that is operably linked to a polynucleotide sequencethat encodes a DNA sequence-specific transactivator; and (ii) a reporterpolynucleotide that is operably linked to a promoter sequence that canbe recognized by the DNA sequence-specific transactivator; wherein thetarget drought-inducible promoter sequence can be recognized by atranscriptional regulatory polypeptide that modulates a signalingpathway and said modulation results in increased tolerance todesiccation in the plant; (b) identifying a test compound that producesa level of induction of the reporter polynucleotide greater than thelevel of induction in a control plant and selecting a hit compound soidentified; and (c) in a secondary screen, contacting the plant with thehit compound and detecting increased tolerance to desiccation in theplant relative to a control plant.
 8. The method of claim 7, wherein thelevel of induction of the reporter polynucleotide is at least 2.5 fold.9. The method of claim 7, wherein the DNA sequence-specifictransactivator is a translational fusion of a DBD comprising a sequenceencoded by SEQ ID NO: 1 and an AD comprising SEQ ID NO:
 20. 10. Themethod of claim 7, wherein the reporter polynucleotide encodes greenfluorescent protein.
 11. The method of claim 7, wherein the targetpromoter sequence is an At3g46230 promoter or a functional part thereofhaving a promoter function.
 12. A method for increasing tolerance todesiccation, performance or yield of a plant, said method comprising thesteps of: (a) contacting at least one test compound with a transgenicplant cell comprising: (i) a target drought-inducible promoter sequencethat is operably linked to a polynucleotide sequence that encodes a DNAsequence-specific transactivator; and (ii) a reporter polynucleotidethat is operably linked to a promoter sequence that can be recognized bythe DNA sequence-specific transactivator; wherein the targetdrought-inducible promoter sequence can be recognized by atranscriptional regulatory polypeptide that modulates specific signalingpathways and said modulation results in increased plant performance oryield in the plant; (b) identifying a test compound that produces alevel of induction of the reporter polynucleotide greater than the levelof induction in a control plant and selecting a hit compound soidentified; and (c) in a secondary screen, contacting the plant with thehit compound and detecting increased tolerance to desiccation,performance or yield in the plant relative to a control plant.
 13. Themethod of claim 12, wherein the level of induction of the reporterpolynucleotide is at least 2.5 fold.
 14. The method of claim 12, whereinthe DNA sequence-specific transactivator is a translational fusion of aDBD comprising a sequence encoded by SEQ ID NO: 1 and an AD comprisingSEQ ID NO:
 20. 15. The method of claim 12, wherein the reporterpolynucleotide encodes green fluorescent protein.
 16. The method ofclaim 12, wherein the target promoter sequence is an At3g46230 promoteror a functional part thereof having a promoter function.